Selective preventives/remedies for progressive lesions after organ damage

ABSTRACT

An object of the present invention is to provide a pharmaceutical agents for preventing and/or treating the progressive lesion after the organic damage without inhibiting organic function or regeneration function thereof, by selectively suppressing the induction of cytotoxic effector macrophages which are induced into the damaged organs in response to chemokines and cytokines which are expressed depending on the type of damaged organic tissues.

TECHNICAL FIELD

[0001] Organs in organism may be damaged, for example, by blood flowdisorder, ischemia reperfusion injury, hypertension, hyperglycemia,hyperlipemia, pharmaceutical agent or viral infection. Damaged organtissues show any of responses of necrosis, natural death orself-regeneration depending upon the degree of the damage. Immune systemdeeply participates in such a response and some of macrophages which arecarriers of such immune participate in self-regeneration of organtissues while others participate in necrosis or natural death of organtissues. The latter, i.e. that which shows cytotoxicity in a functionalmanner among the much differentiated macrophages, is called effectormacrophage and such an effector macrophage further worsens the damageafter the above-mentioned damage and causes a progressive lesion afterthe organic damage.

[0002] The present invention relates to a pharmaceutical in whichinduction of effector macrophage which is a cause of progressive lesionnoted after such an organic damage is selectively suppressed whereby thesaid progressive lesion is prevented and/or treated without inhibitingthe function and regeneration process of the organ, and also relates toa therapeutic method using the same. The present invention furtherrelates to a screening method for compounds which can be the saidpharmaceutical. The present invention still further relates to novelγ-lactone derivatives useful as the said pharmaceutical.

[0003] With regard to one of the progressive lesions noted after theabove-mentioned organic damages, fibrosis of tissue cells may beexemplified. Such a fibrosis of tissue cells is caused by effectormacrophage showing cytotoxic property the same as above. The novelγ-lactone derivatives according to the present invention are able toselectively suppress the induction of the said effector macrophage. As aresult of utilization of such an action, the present invention alsorelates to a pharmaceutical as a fibrosis inhibitor containing the saidnovel γ-lactone derivative.

[0004] Rejection, particularly chronic rejection, upon transplantationof allogenic or xenogenic organ cells is caused by the same mechanism inthe progressive lesion after organic damage or, at least similar,mechanism to that. The novel γ-lactone derivatives according to thepresent invention selectively suppress the induction of effectormacrophage causing the induction of allograft rejection, particularlychronic rejection, upon allogenic or xenogenic cell, tissue or organtransplantation whereby they show an immunosuppressive action only inthe organ tissues showing the rejection. As a result of utilization ofsuch an action, the present invention also relates to a pharmaceuticalas an immunosuppressant containing the said novel γ-lactone derivative.

BACKGROUND OF THE INVENTION

[0005] When an organ in organisms is damaged, for example, by blood flowdisorder, ischemia reperfusion injury, hypertension, hyperglycemia,hyperlipemia, pharmaceutical agent or viral infection, there works anorganism defense mechanism by, for example, T lymphocytes, macrophages,NK cells, fibroblasts, B lymphocytes and antibodies, complements, etc.Particularly, T cells and macrophages have an important participation inthe said organism defense mechanism.

[0006] Damaged organ tissues show any of the responses of necrosis,natural death or self-regeneration depending upon the degree of thedamage. At that time, chemokines, cytokines, etc. are expressed from thesaid tissues to promote self-proliferation and regeneration and, at thesame time, organism defense mechanism of the host is activated as well.It has been presumed that the outcome whether the damaged tissues resultin necrosis or natural death by way of atrophy and fibrosis or theyresult in regeneration of tissues is the result of the interaction ofboth.

[0007] For example, there are several reports for the studies that, inthe damaged renal tissue lesions, the damaged renal tissue cells expresstissue-specific chemokine, cytokine or adhesion factor depending uponthe damaged area, etc. whereby the response is exhibited.

[0008] Wada, et al. reported that, in human crescenticglomerulonephritis, MIP-1α of chemokine is expressed in glomerularcellular crescents in an acute stage while, in interstitial tissues ofthe cases of fibrous crescents in a chronic stage, MCP-1 is expressed(Wada T., Furuichi K., Segawa-Takeda C., Shimizu M., Sakai N., Takeda S.I., Takasawa K., Kida H., Kobayashi K. I., Mukaida N., Ohmoto Y.,Matsushima K., Yokoyama H.: MIP-1α and MCP-1 contribute to crescents andinterstitial lesions in human crescentic glomerulonephritis. KidneyInt., 56: 995-1003, 1999.

[0009] Matsuda, et al. reported that, in model rats suffering fromcrescentic glomerulonephritis, P-secretin and L-secretin are expressedin a discriminated manner in glomerular endothelial cells and ininterstitial tissues from urinary tubule, respectively (MichihiroMatsuda, Kenichi Shikata, Daisuke Ogawa, Shinichi Okada, YasushiShikata, Atsushi Wada. and Hiroshi Makino: Mechanism of Induction ofInfiltration of Leucocytes to Renal Tissues by Secretin, Nippon JinzoGakkaishi, 42: 213, 2000).

[0010] Tesch, et al. reported that, when nephrotoxic serum nephritismodel was prepared in MCP-1 knockout mice and expression of MCP-1 waschecked, the expression of MCP-1 in the damaged site of urinary tubulewas weak as compared with a wild type while the expression of MCP-1 inthe glomerular lesion had no difference from the wild type. They alsoreported that, in the knockout mice where expression of MCP-1 was weak,infiltration of macrophage decreased whereby MCP-1 showed the resultthat it participated in migration of macrophage while, in terms ofdegree of proteinuria in an acute stage, there is no difference betweenboth and there was no relation between tissue damage and macrophageinfiltration in an acute stage (Tesch G. H., Schwarting A., KinoshitaK., Rolins B. J., Kelly V. R.: Monocyte chemoattractant protein-1promotes macrophage-mediated tubular injury, but not glomerular injury,in nephritic serum nephritis, J. Clin. Invest. 1999, 103: 73-80.).

[0011] Not only in kidney but also in exocrine gland and islet ofLangerhans of pancreas, there is noted a difference inchemokine-productive response concerning the lesion after the organtissue damage corresponding to the damaged tissue.

[0012] Cameron, et al. reported that, in the mice where nonobese type Idiabetes was spontaneously occurred, promotion of the expression ofMIP-1α and lowering of the expression of MCP-1 were noted amongchemokines in islets of Langerhans and that there was a correlationbetween damage of islets of Langerhans and onset of diabetes (Cameron M.J., Arreaza G. A., Grattan M., Meagher C., Sharif S., Burdick M. D.,Strieter R. M., Cook D. N., Delovitch T. L.: Differential expression ofCC chemokines and the CCR5 receptor in the pancreas is associated withprogression to type I diabetes, J. Immunol., 2000, 165: 1102-10) .

[0013] In the meanwhile, Andoh, et al. investigated the expression ofchemokines in pancreatitis tissues of human acute pancreatitis cases.They reported that, in acute pancreatitis, expression of MIP-1α was notnoted but expression of MCP-1, IL-8 and RANTES was noted in exocrine,acinar and ductal interstitial pancreas tissues (Andoh A., Takaya H.,Saotome T., Shimada M., Hata K., Araki Y., Nakamura F., Shintani Y.,Fujiyama Y., Bamba T.: Cytokine regulation of chemokine (IL-8, MCP-1,and RANTES) gene expression in human pancreatic periacinarmyofibroblasts, Gastroenterology, 2000, 119: 211-9).

[0014] As mentioned above, it was suggested in human clinical studiesand experimental study models using animals that, in progressive lesionnoted after the organic damage, effector macrophage participated thereincorresponding to the damaged tissues.

[0015] Up to now, therapy of steroids, etc. has been carried out forprogressive lesion after organic damage. However, since steroidalpreparations non-selectively suppress the macrophage, they also suppressthe response of even the macrophage participating in the reaction fortissue regeneration at the same time whereby organism defensivemechanism including the regeneration is lessened. In addition, newtissue damage is induced resulting in lesion and, as a result, there isa problem that effector macrophage mediated by the expression ofchemokines and cytokines is actively induced whereby the inherent lesionis further worsened. As such, the conventional therapy of steroids hasno selectivity in the action, and administration of high dose isnecessary for the therapy of the lesion whereby the side effect isremarkable. In addition, there is a difficulty that a continuous therapyby steroids for a long period is accompanied by a severe side effect.

[0016] On the other hand, like the progressive lesion noted after theabove-mentioned organic damage, organism defense mechanism by T cells,macrophages, etc. participates in rejection in the transplantation oforgan, skin or the like. Until now, there have been known many compoundshaving an immunosuppressive action and, for example, compoundsrepresented by the formula (8)

[0017] (in which R¹ is an optionally substituted phenyl group; R² is anoptionally esterified carboxyl group; and X is oxygen atom or optionallyoxidized sulfur atom) have been known to be useful as a γ-lactoneimmunosuppressant (Japanese Patent Laid-Open No. 04/338,331). However,it is to be still improved so as to show a selective action to targetorgans or tissues or so as to show stronger immunosuppressive action.

DISCLOSURE OF THE INVENTION

[0018] Effector macrophage expresses chemokine receptors or β2 integrinreceptors, etc. corresponding to the lesion inherent to the tissuesafter the organic damage via either T-lymphocyte-independent orT-lymphocyte-dependent response and is selectively induced to andactivated in lesion being mediated by them whereby said effectormacrophage causes a progressive lesion after the tissue damage. Anobject of the present invention is to provide a pharmaceutical in whichexpression and function of chemokine receptors such as CCR2, CCR3 orCCR8, β2 integrin receptors such as CD11b/CD18 or other receptors aresuppressed to selectively suppress the induction of the above-mentionedeffector macrophages whereby progressive lesion after organic damage isprevented and/or treated without inhibiting the function of the organand the regeneration function and also to provide a therapeutic methodusing the same. Since the pharmaceutical according to the presentinvention is able to suppress the induction of effector macrophagescausing the progressive lesion after organic damage in a morelesion-selectively manner, it is possible to use in a high dose and, dueto the selectivity of the said pharmaceutical, undesirable side effectcan be avoided even when it is used in a high dose. Therapy for a longperiod using the said pharmaceutical is now possible as well. Further,when the lesions are different resulting in progressive lesion after theorganic damage in plural tissues, it is possible to carry out moreselective and more effective therapy by combination use of the abovepharmaceuticals showing different selectivity. That is an advantagewhich is not noted in the conventional steroidal agents and knownγ-lactone immunosuppressants having no selectivity. The presentinvention also aims to provide useful and novel γ-lactone derivatives.

[0019] Another object of the present invention is to provide a methodfor screening the compounds which do not substantially suppress themacrophage participating in tissue regeneration and cause theprogressive lesion after the above-mentioned organic damage.

[0020] Still another object of the present invention is to provide amethod for inducing the effector macrophage which causes the progressivelesion after the above-mentioned organic damage.

[0021] Further object of the present invention is to provide apharmaceutical useful as a fibrosis inhibitor preventing from thefibrosis which is one of lesions inherent to the tissues after theorganic damage or, to be more specific, to provide a fibrosis inhibitorcontaining the above-mentioned novel γ-lactone derivative.

[0022] Still further object of the present invention is to provide apharmaceutical which are useful as an immunosuppressant or, to be morespecific, to provide an immunosuppressant which is an immunosuppressantcontaining the above-mentioned novel γ-lactone derivative and shows aselective immunosuppressive action to target organs or tissues unlikethe known γ-lactone immunosuppressants.

[0023] As mentioned above, damaged tissue cells express chemokine,cytokine or adhesion molecule inherent to the tissue. Depending upontype or degree (amount) of the expression or upon defense mechanism oforganism, the expressed chemokine, cytokine, etc. either promoteself-proliferation or regeneration of the damaged tissues or subject thedamaged tissue to necrosis, natural death or degeneration. Here,effector macrophage showing cytotoxic property is selectively inducedcorresponding to type or expressed amount of the said chemokine andcytokine so that the damaged tissues are subjected to necrosis, naturaldeath or degeneration. To be more specific, effector macrophage isselectively induced to the damaged organ tissues (lesion tissues) bychemokine receptors corresponding to the said chemokine expressed in thedamaged organ tissues (lesion tissue) or by β2 integrin, etc.corresponding to a ligand expressed on the said organ tissues (lesiontissues). The induced effector macrophage recognizes the damagedtissues, acts on the said tissues in a cytotoxic manner and induces theprogressive lesion after the organic damage. On the other hand, theeffector macrophage does not recognize the normal tissue whereinchemokine and cytokine which stimulate for inducing and activating thesaid effector macrophage are not expressed and, as a result, the normaltissue is not damaged.

[0024] Accordingly, if induction of effector macrophage in the damagedorgan tissues can be selectively suppressed by way of suppression ofexpression and function of chemokine receptor such as CCR2, CCR3 andCCR8, β2 integrin receptors such as CD11b/CD18 and other receptors,action of the effector macrophage in a cytotoxic manner to the damagedorgan tissues can be prevented and, in addition, induction of themacrophage participating in the tissue regeneration is not suppressed sothat lowering of the defense ability of the organism is not notedwhereby it has been found that progressive lesion after organic damagecan be prevented, mitigated or treated.

[0025] When a compound which is able to selectively suppress theinduction of effector macrophage as such is used as a pharmaceutical,there is reduced the side effect such as that defense of organism isunnecessarily lowered or new tissue damage is induced. In addition, itis possible to result in recovery and regeneration of the tissue withoutan unnecessary lowering of defense of organism and, therefore, it is nowpossible to continuously carry out the therapy for a long period.

[0026] Ishibashi who is one of the inventors of the present inventionhas carried out a further investigation for the progressive lesion inkidney on the basis of the above finding and has obtained an unexpectedfinding that a compound which suppresses the induction of effectormacrophage caused by contact of human peripheral blood mononuclear cellsabbreviated as PBMC with lipopolysaccharide suppresses the progress ofglomerular lesion of kidney and that a compound which suppresses theinduction of effector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC suppresses the progress of progressivelesion of tubulointerstitial tissue after renal damage.

[0027] Ishibashi who is one of the inventors of the present inventionhas also carried out an investigation for progressive lesion in pancreasand obtained an unexpected finding that a compound which suppresses theinduction of effector macrophage caused by contact of human PBMC withlipopolysaccharide suppresses the progress of lesion in islets ofLangerhans of pancreas and that a compound which suppresses theinduction of effector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC suppresses the progress of lesion ofexocrine, acinar and ductal interstitial tissues of pancreas.

[0028] Here, when lesion of islets of Langerhans of pancreas progresses,diabetes mellitus is able to occur. When diabetes mellitus occurs,diabetic nephropathy which is a glomerular lesion is able to occur as acomplication thereof. Ishibashi who is one of the inventors of thepresent invention has further obtained an unexpected finding that acompound which suppresses the induction of effector macrophage caused bycontact of human PBMC with lipopolysaccharide is also able to preventand/or treat the onset of diabetic glomerular lesion (another name:diabetic nephropathy) together with the onset of diabetes mellitus.

[0029] Ishibashi who is one of the inventors of the present inventionhas furthermore obtained an unexpected finding that a compound whichsuppresses the induction of effector macrophage caused by contact ofhuman PBMC with mitomycin-treated human PBMC is also able to preventand/or treat the onset of lesion of tubulointerstitial tissues which isa complication of pancreatitis together with onset of pancreatitis whichis lesion of exocrine, acinar and ductal interstitial tissues ofpancreas.

[0030] The present inventors have carried out an investigation for thecompounds which are able to selectively suppress the induction ofeffector macrophage which is caused corresponding to lesion inherent totissues after the organic damage and, as a result, they have found thatnovel γ-lactone derivatives represented by the following formulae offrom (1) to (7) have such an action.

[0031] The present inventors have further found that, since fibrosis oftissues is one of the progressive lesions after the organic damage, thenovel γ-lactone derivatives represented by the following formulae offrom (1) to (7) are also useful as inhibitors for fibrosis.

[0032] With regard to the rejection at the transplantation of organcells of allogeneic or xenogenic type, there are acute rejection andchronic rejection and it has been known that, particularly in thechronic rejection, not only immunological factors but alsonon-immunological factors such as damage by pharmaceuticals, ischemiareperfusion injury, viral infection, blood flow disorder and exclusionof cells can be a cause. On the other hand, the above-mentionedprogressive lesion after organic damage also results from the organicdamage, which is a trigger, such as damage by pharmaceuticals, ischemiareperfusion injury, viral infection, blood flow disorder and exclusionof cells. Accordingly, chronic rejection is caused by a mechanism whichis the same as or at least similar to the progressive lesion after theorganic damage. Therefore, it has been found that novel γ-lactonederivatives represented by the following formulae of from (1) to (7)which are able to selectively suppress the induction of effectormacrophage induced being correspondent to the lesion inherent to thetissues after organic damage are useful as an immunosuppressant for theprevention or the therapy of rejection upon of allogeneic or xenogeniccell, tissue or organ transplantation, particularly as animmunosuppressant to chronic rejection.

[0033] Thus, the present invention relates to the followings.

[0034] (1) A pharmaceutical composition, which comprises a compoundsuppressing the induction of effector macrophages.

[0035] (2) A preventive and therapeutic pharmaceutical selectively toprogressive lesion of organic damages which comprises a compoundsuppressing the induction of effector macrophages.

[0036] (3) A pharmaceutical for prevention and/or therapy of glomerularlesion of kidney, which comprises a compound suppressing the inductionof effector macrophages caused by contact of human PBMC withlipopolysaccharide.

[0037] (4) A pharmaceutical for prevention and/or therapy of progressivetubulointerstitial lesion after renal damage, which comprises a compoundsuppressing the induction of effector macrophages caused by contact ofhuman PBMC with mitomycin-treated human PBMC.

[0038] (5) A pharmaceutical for prevention and/or therapy of lesion ofislets of Langerhans of pancreas, which comprises a compound suppressingthe induction of effector macrophages caused by contact of human PBMCwith lipopolysaccharide.

[0039] (6) A pharmaceutical for prevention and/or therapy of lesion ofexocrine, acinar and ductal interstitial tissues of pancreas, whichcomprises a compound suppressing the induction of effector macrophagescaused by contact of human PBMC with mitomycin-treated human PBMC.

[0040] (7) A pharmaceutical for prevention and/or therapy of diabetesmellitus and diabetic glomerular lesion, which comprises a compoundsuppressing the induction of effector macrophages caused by contact ofhuman PBMC with lipopolysaccharide.

[0041] (8) A pharmaceutical for prevention and/or therapy ofpancreatitis and lesion of interstitial tissues of urinary tubule whichis a complication of pancreatitis, which comprises a compoundsuppressing the induction of effector macrophages caused by contact ofhuman PBMC with mitomycin-treated human PBMC.

[0042] (9) A method for prevention and/or therapy of glomerular lesionof kidney, lesion of islets of Langerhans of pancreas or diabetesmellitus and diabetic glomerular lesion, which comprises using apharmaceutical containing a compound suppressing the induction ofeffector macrophages caused by contact of human PBMC withlipopolysaccharide.

[0043] (10) A method for prevention and/or therapy of progressivetubulointerstitial lesion after renal damage, lesion of exocrine, acinarand ductal interstitial tissues of pancreas or pancreatitis, and lesionof interstitial tissues of urinary tubule, which comprises using apharmaceutical containing a compound suppressing the induction ofeffector macrophages caused by contact of human PBMC withmitomycin-treated human PBMC.

[0044] (11) A method for screening a compound which is able to prevent,mitigate or treat glomerular lesion of kidney, lesion of islet ofLangerhans of pancreas or diabetes mellitus and diabetic glomerularlesion, which comprises measuring a suppressive action of a compound tobe tested against the induction of effector macrophage caused by contactof human PBMC with lipopolysaccharide.

[0045] (12) The method for screening according to the above-mentioned(11), wherein effector macrophages are induced by incubation of humanuntreated PBMC in RPMI 1640 medium in the presence of a compound to betested, lipopolysaccharide and human AB type serum, the said inducedeffector macrophages are brought into contact with monolayeredautologous erythrocytes and a compound showing less production of SPFC,Spontaneous Plaque-Forming Cell, as compared with the absence of thesaid compound to be tested is screened.

[0046] (13) A method for screening a compound which is able to prevent,mitigate or treat progressive tubulointerstitial lesion after renaldamage, lesion of exocrine acinar and ductal interstitial tissues ofpancreas or pancreatitis and lesion of interstitial tissues of urinarytubule, which comprises measuring a suppressive action of a compound tobe tested against the induction of effector macrophages caused bycontact of human PBMC with mitomycin-treated human PBMC.

[0047] (14) The method for screening according to the above-mentioned(13), wherein effector macrophages are induced by a mixed incubation ofmitomycin-treated human PBMC and human untreated PBMC in RPMI 1640medium in the presence of a compound to be tested and human AB typeserum, the said induced effector macrophages are brought into contactwith monolayered autologous erythrocytes and a compound showing lessproduction of SPFC as compared with the absence of the said compound tobe tested is screened.

[0048] (15) A kit for screening a compound which is able to prevent,mitigate or treat glomerular lesion of kidney, lesion of islet ofLangerhans of pancreas or diabetes mellitus and diabetic glomerularlesion, which comprises (a) human PBMC, (b) lipopolysaccharide, (c)human AB type serum, (d) RPMI 1640 medium and (e) a plate to whichmonolayered autologous erythrocytes are adhered.

[0049] (16) A kit for screening a compound which is able to prevent,mitigate or treat progressive tubulointerstitial lesion after renaldamage, lesion of exocrine, acinar and ductal interstitial tissues ofpancreas or pancreatitis and lesion of interstitial tissues of urinarytubule, which comprises (a) human PBMC, (b) mitomycin-treated humanPBMC, (c) human AB type serum, (d) RPMI 1640 medium and (e) a plate towhich monolayered autologous erythrocytes are adhered.

[0050] (17) A method for the induction of effector macrophages which area cause of glomerular lesion of kidney, lesion of islets of Langerhansof pancreas or diabetes mellitus and diabetic glomerular lesion, whichcomprises bringing lipopolysaccharide into contact with human PBMC.

[0051] (18) A method for the induction of effector macrophages which area cause of progressive tubulointerstitial lesion after renal damage,lesion of exocrine, acinar and ductal interstitial tissues of pancreasor pancreatitis and lesion of interstitial tissues of urinary tubule,which comprises bringing human PBMC into contact with mitomycin-treatedhuman PBMC.

[0052] (19) The pharmaceutical according to any of the above-mentioned(1) to (8), which comprises a compound represented by the formula (1)

[0053] or a compound represented by the formula (2).

[0054] (20) An optical isomer γ-lactone represented by the formula (3)

[0055] or by the formula (4)

[0056] (in the formula, R²¹ is an optionally substituted naphthyl groupand R²² is an optionally substituted straight or branched hydrocarbongroup having 1 to 6 carbon atoms) or a mixture of the above opticalisomers.

[0057] (21) The optical isomer γ-lactone or a mixture of the opticalisomers according to the above-mentioned (20), wherein R²¹ is naphthyland R²² is methyl.

[0058] (22) A compound represented by the formula (5) or apharmacologically acceptable salt thereof.

[0059] (in the formula, (a) R¹ and R² may be the same or different andeach is hydrogen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; X² is O, S orNR³ in which R³ is hydrogen, oxygen, an open-chain aliphatic hydrocarbongroup which may be substituted or interrupted by an intervening group,an optionally substituted cyclic aliphatic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and n is an integer from 1 to 5 or

[0060] (b) x² is O, S or NR³; R¹, R² and R³ each is a substituentrepresented by the formula R¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be thesame or different and each is an optionally substituted open-chain orcyclic hydrocarbon group, an optionally substituted aryl group, anoptionally substituted heterocyclic group or an optionally substitutedcondensed heterocyclic group; and Z is an intervening group); and n isan integer from 1 to 5).

[0061] (23) A compound represented by the formula (6) or apharmacologically acceptable salt thereof.

[0062] (in the formula, R¹, X² and n have the same meaning as defined inthe above-mentioned (4); X¹ is halogen, cyano group, an optionallysubstituted mercapto group, an optionally substituted sulfo group, anoptionally substituted sulfonyl group, an optionally substitutedhydroxyl group, an optionally substituted amino group or an optionallysubstituted phosphoryl group).

[0063] (24) A compound represented by the formula (7) or apharmacologically acceptable salt thereof.

[0064] (in the formula, R¹ and X² have the same meaning as defined inthe above-mentioned (4); X¹ has the same meaning as defined in theabove-mentioned (5); R⁵ and R⁶ may be the same or different and each is(a) hydrogen, a straight or branched aliphatic hydrocarbon group whichmay be substituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, or an optionallysubstituted heterocyclic group, an optionally substituted condensedheterocyclic group,

[0065] (b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in theformula, R¹⁰ and R¹¹ may be the same or different and each is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and Z is an intervening group.), or

[0066] (c) R⁵ and R⁶ together with the carbon atom to which they areattached form an optionally substituted aromatic ring).

[0067] (25) The pharmaceutical according to any of the above-mentioned(1) to (8), which comprises a compound mentioned in any of theabove-mentioned (20) to (24).

[0068] (26) An immunosuppressant or a fibrosis inhibitor which comprisesa compound mentioned in any of the above-mentioned (19) to (24).

[0069] (27) The pharmaceutical according to any of the above-mentioned(1) to (8), which comprises ethyl 2-ketoglutarate or benzyl2-ketoglutarate.

BEST MODE FOR CARRYING OUT THE INVENTION

[0070] (1) Firstly, the present invention provides a method for theinduction of effector macrophage which is induced and activatedcorresponding to the lesion inherent to the tissues after the organicdamage and results in progressive lesion after the organic damage.

[0071] Thus, the present invention provides (a) a method for theinduction of effector macrophage which is a cause of glomerular lesionof kidney, lesion of islets of Langerhans of pancreas or diabetesmellitus and diabetic glomerular lesion, characterized in that,lipopolysaccharide is brought into contact with human PBMC (hereinafter,abbreviated as “LPS inducing method”) and (b) a method for the inductionof effector macrophage which is a cause of progressivetubulointerstitial lesion after renal damage, lesion of exocrine, acinaror ductal interstitial tissues of pancreas or pancreatitis andtubulointerstitial lesion, characterized in that, human PBMC are broughtinto contact with mitomycin-treated human PBMC (hereinafter, abbreviatedas “allo-MLC inducing method”).

[0072] Hereunder, an LPS inducing method will be illustrated in detail.

[0073] With regard to a lipopolysaccharide used for the LPS inducingmethod, that which has been known per se may be used. For example, thelipopolysaccharide derived from Gram-negative bacteria such asSalmonella and Escherichia coli may be used. It may be either theso-called rough type or smooth type.

[0074] For the preparation of the lipopolysaccharide, a method which hasbeen known per se may be used. An example is a method where it isextracted from microbe and, if desired, a treatment for removing thetoxicity is conducted. Examples of the method for extracting frommicrobe are a method of extracting with hot phenol (Westphal & Jann.,Methods Carbohydr. Chem., 5, 83-99 (1965)) and a method where microbe istreated with proteinase K in the presence of sodium lauryl sulfate. Inaddition, chemically synthesized one may be used or the commerciallyavailable one may be appropriately used.

[0075] In the present invention, it is preferred that lipopolysaccharideis used as a solution and, when it is made into a solution using anappropriate solvent, preferably, RPMI 1640 liquid, it is preferred touse a solution of a high concentration of about 60 to 100 μg/ml, morepreferably about 70 to 90 μm/ml or still more preferably about 80 μg/ml.

[0076] The human PBMC can be obtained from human peripheral blood by amethod known per se. An example for a method for separating themononuclear cells from human peripheral blood is a method by acentrifugal separation using Ficoll-Paque (registered trademark;manufactured by Pharmacia Fine Chemicals). To be more specific, theabove-mentioned method comprises (a) a step where Ficoll-Paque is placedat the bottom of a test tube, (b) a step where a blood sample as it isor after being diluted is carefully transferred onto the Ficoll-Paqueusing a pipette, (c) a step where the blood preparation prepared byFicoll-Paque in (b) is centrifuged at about 400˜500 G for about 30˜40minutes so that a blood component having larger specific gravity thanthe specific gravity of Ficoll-Paque comes into Ficoll-Paque or passesFicoll-Paque and (d) a step where the mononuclear cell layer separatedon the upper area of Ficoll-Paque is collected.

[0077] A specific mode for the LPS inducing method is a method wherehuman PBMC is incubated in RPMI 1640 medium in the presence oflipopolysaccharide and human AB type serum to induce the effectormacrophage and that method is advantageously used in the presentinvention.

[0078] At that time, a combination of any two of or all oflipopolysaccharide, human AB type serum and human PBMC may be previouslymixed and then added to the medium or each of them may be added to themedium solely. There is no limitation for the order of adding to themedium. More preferably however, human untreated PBMC are added to RPMI1640 medium to which human AB type serum is previously added and thenlipopolysaccharide is added thereto.

[0079] RPMI 1640 medium is mentioned in Goding, J. W. (1980) J. Immunol.Methods, 39, 185 and JAMA, 199 (1957), 519. Alternatively, acommercially available product (manufactured by Sigma) may be used aswell.

[0080] More preferred mode for carrying out the LPS inducing method isas follows.

[0081] Human untreated PBMC are dissolved in a concentration of about2×10⁶ cells/ml to RPMI 1640 medium to which gentamicin, L-glutamine andhuman AB type serum are added to concentrations of about 5 μg/ml, about2 mM and about 10% by weight, respectively and then lipopolysaccharideis added thereto so as to make its final concentration about 80 μg/ml toprepare a culture liquid. The said culture liquid is incubated at about37° C. for about six days with about 5% of CO₂.

[0082] A method for the induction of allo-MLC will be mentioned indetail as follows.

[0083] The mitomycin-treated human PBMC used in an allo-MLC inducingmethod can be prepared, for example, by adding mitomycin to the humanPBMC obtained by the above-mentioned known method to make the finalconcentration of mitomycin about 40 μg/ml followed by subjecting to aheating treatment at about 37° C. for about 30 minutes.

[0084] A specific mode for an allo-MLC inducing method is a method wheremitomycin-treated human PBMC and normal human untreated PBMC aresubjected to a mixed culture in RPMI 1640 medium in the presence ofhuman AB type serum to induce the effector macrophage and that method isadvantageously used in the present invention.

[0085] At that time, a combination of any two of or all of human AB typeserum, mitomycin-treated human PBMC and human PBMC may be previouslymixed and then added to the medium or each of them may be added to themedium solely. There is no limitation for the order of adding to themedium. It is preferred however that normal human untreated PBMC areadded to RPMI 1640 medium to which human AB type serum is previouslyadded and then mitomycin-treated human PBMC are added thereto.

[0086] A more preferred mode of carrying out the allo-MLC inducingmethod will be as follows.

[0087] Human untreated PBMC are dissolved in a concentration of about2×10⁶ cells/ml to RPMI 1640 medium to which gentamicin, L-glutamine andhuman AB type serum are added to concentrations of about 5 μg/ml, about2 mM and about 10% by weight, respectively and then mitomycin-treatedhuman PBMC are added thereto so as to make the final concentration ofabout 2×10⁶ cells/ml to prepare a culture liquid. The said cultureliquid is incubated at about 37° C. for about six days with about 5% ofCO₂.

[0088] (2) The present invention then provides a method for screening acompound which is able to prevent, mitigate or treat the progressivelesion after organic damage by a selective suppression of induction ofeffector macrophage corresponding to lesion inherent to the tissuesafter organic damage.

[0089] Thus, the present invention provides a method for screening acompound which is able to prevent, mitigate or treat glomerular lesionof kidney, lesion of islets of Langerhans of pancreas or diabetesmellitus and diabetic glomerular lesion, characterized in that, asuppressive action of a compounded to be tested to the induction ofeffector macrophage caused by contact of human PBMC withlipopolysaccharide is measured.

[0090] Preferred embodiment of the above-mentioned screening methodaccording to the present invention will be as follows.

[0091] First, human PBMC are brought into contact withlipopolysaccharide in the presence of the compound to be tested toinduce effector macrophage. Then, measurement is carried out to checkwhether the compounded to be tested shows a suppressive action toinduction of effector macrophage.

[0092] It is preferred that the above-mentioned induction of effectormacrophage is carried out in the same manner as in the above-mentionedLPS inducing method except that, when lipopolysaccharide is brought intocontact with human PBMC, the compound to be tested is further present.

[0093] It is preferred that the measurement of the above-mentionedsuppressive action to the induction of effector macrophage is carriedout by measuring the numbers of spontaneous plaque-forming cells(hereinafter, referred to as SPFC) which are produced by bringing theinduced effector macrophage into contact with monolayered autologouserythrocytes. Thus, when the numbers of the produced SPFC are less thanthe case where no test compound to be tested is present, it is judgedthat induction of effector macrophage is suppressed.

[0094] More preferred embodiment will be as follows.

[0095] Human untreated PBMC are dissolved in a concentration of about2×10⁶ cells/ml to RPMI 1640 medium to which gentamicin, L-glutamine andhuman AB type serum are added to concentrations of about 5 μg/ml, about2 mM and about 10% by weight, respectively and then lipopolysaccharideis added to the solution so as to make its final concentration of about80 μg/ml.

[0096] A compound to be tested is dissolved in an appropriate solvent,preferably dimethyl sulfoxide (DMSO) of about 0.001% by weight and theresulting test solution is added to above-mentioned medium. At thattime, there are prepared various media where concentrations of thecompound to be tested are different. It is preferred that theconcentrations of the compound to be tested are about 1 μM to 0.001 μM.

[0097] The mixture is incubated at about 37° C. for about six days inthe presence of about 5% of CO₂ to induce effector macrophage. Theeffector macrophage generated in the cultured PBMC induced from theculture liquid is recovered. In the recovering, known methods may beused and, for example, there is a method where the adhered thing isrecovered using a rubber-policeman (spatula made of rubber). After that,washing may be carried out. For the washing, it is preferred to use aHanks solution to which gentamicin is added so as to make itsconcentration of about 5 μg/ml.

[0098] In the meanwhile, monolayered autologous erythrocytes areprepared. The monolayered autologous erythrocytes may be manufactured bymethods known per se but the following method is preferred.

[0099] Autologous erythrocytes are made about 4% by weight concentrationby a Hanks solution without serum supplementation. It is preferred touse the autologous erythrocytes which are prepared by theabove-mentioned known method and preserved at about 4° C. in aphosphate-buffered physiological saline (hereinafter, referred to asPBS) with addition of 0.1% of AB serum. Poly-L-lysine is added to aTerasaki plate, treated at about 37° C. for about 20 minutes and washedwith PBS, the above-mentioned autologous erythrocytes are addedimmediately thereafter and allowed to stand at about 37° C. for about 30minutes and the erythrocytes which are not adhered are removed to give aTerasaki plate to which monolayered autologous erythrocytes are adhered.

[0100] The induced effector macrophage which is recovered hereinabove isdissolved/suspended in the Hanks solution with 5 μg/ml of gentamicin soas to make about 2×10⁶ cells/ml. To the above Terasaki plate to whichthe monolayered autologous erythrocytes are adhered is added 1 to 10 μlof the Hanks solution, the above dissolved/suspended liquid is addedthereto in the same amount and the mixture is allowed to stand at about37° C. for about 2 hours. After completion of the reaction, it ispreferred to fix by formalin.

[0101] Numbers of the produced SPFC can be easily measured by aphase-contrast microscope.

[0102] With regard to an index for the suppression of induction ofeffector macrophage, it is preferred to use an IC₅₀ concentration. TheIC₅₀ can be calculated as follows.

[0103] When plural experiments are carried out under the sameconditions, a mean value of the SPFC production numbers measured asabove is determined. Then, SPFC production numbers per 1×10⁶ of inducedmacrophages are calculated and, from the recovered induced macrophagenumbers, the produced SPFC numbers are determined (this value will becalled S1). Effector macrophage is induced as above without addition ofthe compound to be tested and SPFC production numbers are measured bythe same manner (this value will be called S2). Concentration of thesubstance to be tested when Si becomes one half of S2 is defined asIC₅₀.

[0104] With regard to the compound which is able to prevent, mitigate orcure the progressive lesion after organic damage found by the screeningmethod according to the present invention, the compound where IC₅₀ is 1μM or less is preferred.

[0105] The present invention further provides a method for screening acompound which is able to prevent, mitigate or treat progressivetubulointerstitial lesion after renal damage, lesion of exocrine, acinaror ductal interstitial tissues of pancreas or pancreatitis, and lesionof tubulointerstitial tissues associated with pancreatitis,characterized in that, a suppressive action of a compounded to be testedto the induction of effector macrophage caused by contact of human PBMCwith mitomycin-treated human PBMC is measured.

[0106] A preferred embodiment of the above-mentioned screening methodaccording to the present invention is as follows. Firstly, human PBMCare brought into contact with mitomycin-treated human PBMC in thepresence of a compound to be tested to induce effector macrophage. Afterthat, a measurement is carried out to check whether the compound to betested shows a suppressive action to the induction of effectormacrophage.

[0107] It is preferred to induce the above effector macrophage in thesame manner as in the case of inducing method for the above allo-MLCexcept that the compound to be tested is further present in bringing thehuman PBMC into contact with mitomycin-treated human PBMC. Concentrationof the compound to be tested is preferably in about 1 μM to 0.001 μM thesame as in the above screening method and it is preferred to conduct theinduction of effector macrophage by adding the compounded to be testedin various concentrations.

[0108] It is preferred that the measurement of the suppressive action ofthe compound to be tested to induction of the above-mentioned effectormacrophage is carried out by the same manner as in the above-mentionedscreening method.

[0109] The above-mentioned compound to be tested may be anything and maybe, for example, peptide, protein, non-peptidic compound, syntheticcompound, fermented product, cell extract, vegetable extract, animaltissue extract and plasma. It may be either a known compound or a novelcompound.

[0110] It is also possible to combine known methods in such a mannerthat plural compounds to be tested are screened at the same time and,only when a suppressive action to the induction of effector macrophageis detected, suppressive action for each compound is measured by theabove-mentioned method and the compound having a suppressive action isidentified, etc.

[0111] The present invention further provides a screening kit forcarrying out the above-mentioned screening method. There is noparticular limitation for the form of the said screening kit althoughthe forms which have been known per se may be used.

[0112] For example, a preferred embodiment of a screening kit forscreening a compound which is able to prevent, mitigate or treatglomerular lesion of kidney, lesion of islets of Langerhans of pancreasor diabetes mellitus and diabetic glomerular lesion is a kit whichcomprises (a) human PBMC, (b) lipopolysaccharide, (c) human AB typeserum, (d) RPMI 1640 medium and (e) a plate to which monolayeredautologous erythrocytes are adhered.

[0113] Preferred one is such a kit containing Hanks solution to which(a) a solution prepared by adding human PBMC and lipopolysaccharide areadded to an extent of about 2×10⁶/ml and about 80 μg/ml, respectively toRPMI 1640 containing about 5 μg/ml of gentamicin, about 2 mM ofL-glutamine and about 10% by weight of human AB type serum, (b) a plateto which monolayered erythrocytes are adhered and (c) about 5 μg/ml ofgentamicin are added.

[0114] A preferred embodiment of a screening kit for screening acompound which is able to prevent, mitigate or treat progressivetubulointerstitial lesion after renal damage, or lesion of exocrine,acinar or ductal interstitial tissues of pancreas is a kit comprising(a) human PBMC, (b) mitomycin-treated human PBMC, (c) human AB typeserum, (d) RPMI 1640 medium and (e) a plate to which monolayeredautologous erythrocytes are adhered.

[0115] Preferred one is the said kit containing a Hanks solution towhich (a) a solution prepared by adding human PBMC and mitomycin-treatedhuman PBMC to an extent of about 2×10⁶/ml each to RPMI 1640 containingabout 5 μg/ml of gentamicin, about 2 mM of L-glutamine and about 10% byweight of human AB type serum, (b) a plate to which monolayerederythrocytes are adhered and (c) about 5 μg/ml of gentamicin are added.

[0116] (3) The present invention provides a pharmaceutical forprevention or therapy of progressive lesion after organic damage by aselective suppression of induction of effector macrophage correspondingto the lesion inherent to the tissues after the organic damage and alsoprovides a therapeutic method using the said pharmaceutical.

[0117] Thus, the present invention provides a pharmaceutical forprevention and/or therapy of glomerular lesion of kidney, lesion ofislets of Langerhans of pancreas or diabetes mellitus or diabeticglomerular lesion, characterized in that, the pharmaceutical contains acompound which suppresses the induction of effector macrophage caused bycontact of human PBMC with lipopolysaccharide.

[0118] The present invention also provides a pharmaceutical forprevention and/or therapy of progressive tubulointerstitial lesion afterrenal damage, lesion of exocrine, acinar or ductal interstitial tissuesof pancreas and pancreatitis and lesion of tubulointerstitial tissueswhich is a complication of pancreatitis, characterized in that, thepharmaceutical contains a compound which suppresses the induction ofeffector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC.

[0119] With regard to (a) a compound which suppresses the induction ofeffector macrophage caused by contact of human PBMC withlipopolysaccharide and (b) a compound which suppresses the induction ofeffector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC, the compounds which show the suppressionto the induction of effector macrophage by the above-mentioned screeningmethods may be exemplified.

[0120] When a compound which shows activity as a result of the screeningis acidic or basic, a salt of such a compound may be used as thepharmaceutical as well. Salt of the said compound is a salt with aphysiologically acceptable acid (such as inorganic acid and organicacid) or base (such as alkaline metal). To be more specific, there maybe used inorganic acid salt such as a salt with hydrochloric acid,phosphoric acid, hydrobromic acid and sulfuric acid; organic acid saltsuch as a salt with acetic acid, formic acid, propionic acid, fumaricacid, maleic acid, succinic acid, tartaric acid, citric acid, malicacid, oxalic acid, benzoic acid, methanesulfonic acid andbenzenesulfonic acid; inorganic base salt such as sodium salt, potassiumsalt and ammonium salt; and organic base salt such as dimethylamine saltand cyclohexylamine salt.

[0121] The compound which is used for a pharmaceutical or a therapeuticmethod according to the present invention may be a prodrug or aderivative of the above-mentioned compound as well.

[0122] With regard to the compound used as a pharmaceutical according tothe present invention, the following compounds may be specificallyexemplified.

[0123] Specific examples of the compound which is able to prevent,mitigate or cure glomerular lesion of kidney, lesion of islet ofLangerhans of pancreas or diabetes mellitus or diabetic glomerularlesion by suppressing the induction of effector macrophage caused bycontact of human PBMC with lipopolysaccharide are the followingcompounds (9-1), (7-3), (7-5) and (4-2).

[0124] Specific examples of the compounds which is able to prevent,mitigate or cure progressive tubulointerstitial lesion after renaldamage, lesion of exocrine, acinar or ductal interstitial tissues ofpancreas and pancreatitis and lesion of tubulointerstitial tissues whichis a complication of pancreatitis by suppressing the induction ofeffector macrophage caused by contact of human PBMC withmitomycin-treated human PBMC are the following compounds (6-1), (6-2)and (3-2).

[0125] With regard to the above-mentioned compounds, there are acompound represented by the formula (1)

[0126] and a compound represented by the formula (2).

[0127] There is also an optical isomer γ-lactone represented by theformula (3)

[0128] or by the formula (4)

[0129] (in the formulae, R²¹ is an optionally substituted naphthyl andR²² is an optionally substituted open-chain hydrocarbon group having 1to 6 carbons) and a mixture of such optical isomers.

[0130] Among them, the compound where R²¹ is naphthyl group and R²² ismethyl group is preferred.

[0131] There is also a compound represented by the formula (5) and apharmacologically acceptable salt thereof.

[0132] (in the formula,

[0133] (a) R¹ and R² may be the same or different and each is hydrogen,an open-chain aliphatic hydrocarbon group which may be substituted orinterrupted by an intervening group, an optionally substituted cyclicaliphatic hydrocarbon group, an optionally substituted aryl group, anoptionally substituted heterocyclic group or an optionally substitutedcondensed heterocyclic group; X² is O, S or NR³ in which R³ is hydrogen,oxygen, an open-chain aliphatic hydrocarbon group which may besubstituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and n is aninteger from 1 to 5 or

[0134] (b) X² is O, S or NR³; R¹, R² and R³ each is a substituentrepresented by the formula R¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be thesame or different and each is an optionally substituted open-chain orcyclic hydrocarbon group, an optionally substituted aryl group, anoptionally substituted heterocyclic group or an optionally substitutedcondensed heterocyclic group; and Z is an intervening group); and n isan integer from 1 to 5).

[0135] There is further a compound represented by the formula (6) or apharmacologically acceptable salt thereof.

[0136] (in the formula, R¹, X² and n have the same meaning as defined inthe above-mentioned item(4); X¹ is halogen, cyano group, an optionallysubstituted mercapto group, an optionally substituted sulfo group, anoptionally substituted sulfonyl group, an optionally substitutedhydroxyl group, an optionally substituted amino group or an optionallysubstituted phosphoryl group).

[0137] There is still further a compound represented by the formula (7)or a pharmacologically acceptable salt thereof.

[0138] (in the formula, R¹ and X² have the same meaning as defined inthe above-mentioned item (4); X¹ has the same meaning as defined in theabove-mentioned item(5); R⁵ and R⁶ may be the same or different and eachis

[0139] (a) hydrogen, an open-chain aliphatic hydrocarbon group which maybe substituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted heterocyclic group or an optionally substituted condensedheterocyclic group,

[0140] (b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in theformula, R¹⁰ and R¹¹ may be the same or different and each is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and Z is an intervening group.), or

[0141] (c) R⁵ and R⁶ is an optionally substituted aromatic ring togetherwith the carbon atom to which they are attached).

[0142] In the compound represented by the formula (3) or (4), R²¹ is anoptionally substituted naphthyl group.

[0143] Examples of the substituent are halogen (preferably, fluorine,chlorine and bromine), an oxo group, an alkanoyl group (preferablyC₁₋₈), an alkanoyloxy group (preferably C₁₋₈), an alkanoylamino group(preferably C₁₋₈), carboxyl group, an alkoxycarbonyl group (preferablyC₂₋₈), a haloalkylcarbonyl group (preferably C₂₋₈), an alkoxy group(preferably C₁₋₈), a haloalkoxy group (preferably C₁₋₈), amino group, analkylamino group (preferably C₁₋₈), a dialkylamino group (preferablyC₂₋₁₆), a cyclic amino group, an alkylaminocarbonyl group (preferablyC₂₋₈), carbamoyl group, hydroxyl group, nitro group, cyano group,mercapto group, an alkylthio group (preferably C₁₋₈), analkylsulfonyloxy group (preferably C₁₋₈), an alkylsulfonylamino group(preferably C₁₋₈) and phenyl group.

[0144] Further, the naphtyl group may be substituted by such group(s) atone or more position(s).

[0145] The naphthyl group represented by a substituent R²¹ may also besubstituted with open-chain or cyclic hydrocarbon group which will bementioned later in detail. Carbon number(s) of the hydrocarbon groupis/are preferably 1 to 8. The said open-chain or cyclic hydrocarbongroup may be substituted, for example, with halogen, hydroxyl group,amino group, nitro group, cyano group, mercapto group, carbamoyl group,alkanoyl group, alkanoyloxy group or alkanoylamino group.

[0146] The open-chain hydrocarbon group as a substituent for naphthylgroup may be interrupted by an intervening group such as —O—, —CO—,—COO—, —S—, —SO—, —SO₂—, —NH—, —NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—,—NR³—SO₂—, —Si— or phosphoryl group.

[0147] The substituent R³ is

[0148] (a) hydrogen, oxygen, an open-chain hydrocarbon residue which maybe substituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group or

[0149] (b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in whichR¹⁰ and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group).

[0150] The naphthyl group represented by the substituent R²¹ may besubstituted with a substituent represented by R¹⁰—Z—R¹¹— (in theformula, R¹⁰ and R¹¹ may be the same or different and each is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and Z is an intervening group).

[0151] In the compound represented by the formula (3) or (4), R²² is anoptionally substituted open-chain hydrocarbon group having 1 to 6carbon(s).

[0152] The term “open-chain hydrocarbon group having 1 to 6 carbon(s)”means that carbon number(s) therein is/are 1 to 6 and it may be eitherstraight or branched and either saturated or unsaturated.

[0153] Its examples are methyl group, ethyl group, propyl group,isopropyl group, n-butyl group, isobutyl group, tert-butyl group,n-pentyl group, isopentyl group, tert-pentyl group and n-hexyl group.

[0154] Examples of the substituent in the open-chain hydrocarbon groupare halogen, hydroxyl group, amino group, nitro group, cyano group,mercapto group, carbamoyl group, alkanoyl group, alkanoyloxy group andalkanoylamino group.

[0155] In the compounds represented by the formulae (5) to (7), thesubstituents R¹ and R² may be the same or different and each is

[0156] (a) hydrogen, an open-chain aliphatic hydrocarbon group which maybe substituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group or

[0157] (b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in whichR¹⁰ and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group).

[0158] The term “open-chain aliphatic hydrocarbon group” in thesubstituents R¹ and R² may be straight or branched and may be saturatedor unsaturated.

[0159] To be more specific, there may be exemplified an alkyl group suchas methyl group, ethyl group, n-propyl group, isopropyl group, n-butylgroup, isobutyl group, tert-butyl group, n-pentyl group, isopentylgroup, tert-pentyl group, n-hexyl group, 1,1-dimethylpropyl group and3-methyl-3-butenyl group; an alkenyl group such as vinyl group, allylgroup, 1-propenyl group, isopropenyl group, 2-butenyl group,1,3-butadienyl group and 2-pentenyl group; and an alkynyl group such asethynyl group, 2-propynyl group, 1-butynyl group and 2-butynyl group.

[0160] It is also possible that both double bond and triple bond arepresent in one substituent such as 2-penten-4-ynyl.

[0161] Carbon number(s) is/are preferably 1 to 8. Especially for thesubstituent R², ethynyl group or 2-propynyl group are preferred.

[0162] The “cyclic aliphatic hydrocarbon group” in the substituent R¹and R² may be saturated or unsaturated or may be cross-linked.

[0163] To be more specific, there may be exemplified a cycloalkyl groupsuch as cyclopropyl group, cyclobutyl group, cyclopentyl group,cyclohexyl group, cycloheptyl group, adamantyl group andbicyclo[2.2.1]heptyl group; and a cycloalkenyl group such as2-cyclopenten-1-yl group and 2,4-cyclopentadien-1-yl group.

[0164] Carbon numbers of the cyclic aliphatic hydrocarbon group arepreferably 3 to 12.

[0165] The open-chain or cyclic aliphatic hydrocarbon group in thesubstituents R¹ and R² may be substituted with the substituent whichwill be mentioned later. With regard to the position of such asubstituent, there is no particular limitation so far as it ischemically allowed.

[0166] The open-chain hydrocarbon group as a substituent in thesubstituents R¹ and R² may be interrupted by an intervening group suchas —O—, —CO—, —COO—, —S—, —SO—, —SO₂—, —NH—, —NR³—, —NH—CO—, —NR³—CO—,—NH—SO₂—, —NR³—SO₂—, —Si— or phosphoryl group. R³ is

[0167] (a) hydrogen, oxygen, an open-chain hydrocarbon residue which maybe substituted or interrupted by an intervening group, an optionallysubstituted cyclic aliphatic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group or

[0168] (b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in whichR¹⁰ and R¹¹may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocyclic group; and Z is anintervening group).

[0169] The “aryl group” in the substituents R¹ and R² is an aromatichydrocarbon group which may be partially saturated and there may beexemplified phenyl group, benzyl group, biphenyl group, indenyl groupand naphthyl group or a partially saturated group thereof such as2,3-dihydroindenyl group and 1,2,3,4-tetrahydronaphthyl group.

[0170] Preferred carbon numbers of the aryl group are 6 to 20.

[0171] Such an aryl group may be substituted with the substituent whichwill be mentioned later and, with regard to the position of the linkageand the position of the substituent, there is no particular limitationso far as they are chemically allowed.

[0172] With regard to the substituent R¹ and R², benzyl group ispreferred and, with regard to the substituent R², it is also preferredthat 4-position is substituted with fluorine.

[0173] With regard to the “heterocyclic group” in the substituent R¹ andR², there may be exemplified five- to six-membered saturated orunsaturated ring containing 1 to 3 hetero atom(s) selected from nitrogenatom, oxygen atom and sulfur atoms in a ring.

[0174] Examples of such a heterocyclic group are an aromaticheterocyclic group such as pyrrolyl group, furyl group, thienyl group,imidazolyl group, oxazolyl group, thiazolyl group, pyrazolyl group,isoxazolyl group, isothiazolyl group, oxadiazolyl group, triazolylgroup, indolyl group, benzofuryl group, benzothienyl group,benzimidazolyl group, benzoxazolyl group, benzothiazolyl group, pyridylgroup, pyrimidinyl group, pyrazinyl group, pyridazinyl group, triazolylgroup, tetrazolyl group, quinolyl group and isoquinolyl group; apartially saturated heterocyclic group such as pyranyl group,1,2-dihydroquinolyl group, 1,2,3,4-tetrahydroquinolyl group,1,2-dihydroisoquinolyl group, 1,2,3,4-tetrahydroisoquinolyl group,dihydrofuryl group and dihydrothienyl group; and a saturatedheterocyclic group such as pyrrolidinyl group, piperidinyl group,piperazinyl group, morpholinyl group, tetrahydrofuryl group andtetrahydrothienyl group.

[0175] Such a heterocyclic group may be substituted with the substituentwhich will be mentioned later and, with regard to the position of thelinkage and the position of the substituent, there is no particularlimitation so far as they are chemically allowed.

[0176] With regard to the “condensed heterocyclic group” in thesubstituent R¹ and R², there may be exemplified a case where a five- tosix-membered saturated, partially saturated or unsaturated ringcontaining 1 to 3 hetero atom(s) selected from nitrogen atom, oxygenatom and sulfur atom in the ring is condensed with a benzene ring orother heterocyclic ring.

[0177] With regard to the condensed heterocyclic ring, there may beexemplified indole, 3H-indole, isoindole, benzofuran, benzothiophene,1H-indazole, benzimidazole, benzoxazole, benzothiazole, benzisodxazole,benzisothiazole, quinoline, isoquinoline, quinazoline,1,2-dihydroquinoline, 1,2,3,4-tetrahydroquinoline,1,2-dihydroisoquinoline and 1,2,3,4-tetrahydroisoquinoline.

[0178] Such a condensed heterocyclic group may be substituted with thesubstituent which will be mentioned later. With regard to the positionof the linkage and the position of the substituent in case substituentis present, there is no particular limitation so far as they arechemically allowed.

[0179] The substituents R¹ and R²may be a substituent represented by theformula R¹⁰—Z—R¹¹—.

[0180] R¹⁰ and R¹¹ may be the same or different and each is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed hetero group.With regard to the substituent, there may be exemplified a substituentwhich will be mentioned later.

[0181] With regard to the “substituent” in the open-chain or cyclicaliphatic hydrocarbon group, the aryl group, the heterocyclic group orthe condensed heterocyclic group, a substituent which is conventionallyused in the field of pharmaceuticals may be used.

[0182] Examples of the substituent are halogen (preferably, fluorine,chlorine and bromine), an oxo group, an alkanoyl group (preferablyC_(1˜8)), an alkanoyloxy group (preferably C_(1˜8)), an alkanoylaminogroup (preferably C_(1˜8)), carboxyl group, an alkoxycarbonyl group(preferably C_(2˜8)), a haloalkylcarbonyl group (preferably C_(2˜8)), analkoxy group (preferably C_(1˜8)), a haloalkoxy group (preferablyC_(1˜8)), an alkyl group (preferably C_(1˜20)), amino group, analkylamino group (preferably C_(1˜8)), a dialkylamino group (preferablyC_(2˜16)), a cyclic amino group, an alkylaminocarbonyl group (preferablyC_(2˜8)), carbamoyl group, hydroxyl group, nitro group, cyano group,mercapto group, an alkylthio group (preferably C_(1˜8)), analkylsulfonyloxy group (preferably C_(1˜8)), an alkylsulfonylamino group(preferably C_(1˜8)) and phenyl group.

[0183] Further, the group may be substituted by such group(s) at one ormore position(s).

[0184] The substituent X² is O, S, NH or NR³. Here, the substituent R³has the same meaning as defined above.

[0185] n is an integer of 1 to 5 and, preferably, it is 1.

[0186] The substituent X¹ is (a) halogen, (b) cyano group, (c) anoptionally substituted mercapto group, an optionally substituted sulfogroup or an optionally substituted sulfonyl group, (d) an optionallysubstituted hydroxyl group, (e) an optionally substituted amino group or(f) an optionally substituted phosphoryl group.

[0187] Examples of the substituent are halogen (preferably, fluorine,chlorine and bromine), an oxo group, an alkanoyl group (preferablyC_(1˜8)), an alkanoyloxy group (preferably C_(1˜8)), an alkanoylaminogroup (preferably C_(1˜8)), carboxyl group, an alkoxycarbonyl group(preferably C_(2˜8)), a haloalkylcarbonyl group (preferably C_(2˜8)), analkoxy group (preferably C_(1˜8)), a haloalkoxy group (preferablyC_(1˜8)), an alkyl group (preferably C_(1˜20)), amino group, analkylamino group (preferably C_(1˜8)), a dialkylamino group (preferablyC_(2˜16)), a cyclic amino group, an alkylaminocarbonyl group (preferablyC_(2˜8)), carbamoyl group, hydroxyl group, nitro group, cyano group,mercapto group, an alkylthio group (preferably C_(1˜8)), analkylsulfonyloxy group (preferably C_(1˜8)), an alkylsulfonylamino group(preferably C_(1˜8)) and phenyl group.

[0188] Further, the group may be substituted by such group(s) at one ormore position(s), if chemically acceptable.

[0189] With regard to the above-mentioned optionally substitutedmercapto group, optionally substituted sulfo group or optionallysubstituted sulfonyl group, there may be exemplified benzoylthio group,tosyl group, phenylsulfo group and phenylsulfinyl group.

[0190] With regard to the above-mentioned optionally substitutedhydroxyl group, there may be exemplified methoxy, ethoxy, propionyloxy,allyloxy, benzoxy and naphthoxy.

[0191] With regard to the above-mentioned optionally substituted aminogroup, there may be exemplified methylamino group, ethylamino group,anilino group and anisidino group.

[0192] With regard to the above-mentioned optionally substitutedphosphoryl group, a substituent represented by the formula (9)

[0193] or by the formula (10)

[0194] (in the formulae, substituents R⁷ and R⁸ may be the same ordifferent and each is an optionally substituted open-chain or cyclichydrocarbon group, an optionally substituted aryl group, an optionallysubstituted heterocyclic group or an optionally substituted condensedhetero group) is preferred. There may be exemplified methylphosphorylgroup, dimethylphosphoryl group and methylethylphosphoryl group. Carbonnumber(s) of the said phosphoryl group is/are preferably 1 to 20.

[0195] In the compound (7), the substituents R⁵ and R⁶ each is (a)hydrogen, an open-chain hydrocarbon residue which may be substituted orinterrupted by an intervening group, an optionally substituted cyclicaliphatic hydrocarbon group, an optionally substituted aryl group, anoptionally substituted heterocyclic group or an optionally substitutedcondensed heterocyclic group or is (b) a substituent represented by theformula R¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be the same or differentand each is an optionally substituted open-chain or cyclic hydrocarbongroup, an optionally substituted aryl group, an optionally substitutedheterocyclic group or an optionally substituted condensed heterocyclicgroup; and Z is an intervening group).

[0196] To be specific, the above is the same description as that for thesubstituents R¹ and R².

[0197] The substituents R⁵ and R⁶ may be the same or different.

[0198] The substituents R⁵ and R⁶ may also form an aromatic ringtogether with a carbon to which they are bonded. There are exemplifiedthe cases where benzene, naphthalene or indene is formed.

[0199] The said aromatic ring may be substituted. In that case, one ormore position(s) may be substituted. Examples of the substituent arehalogen (preferably, fluorine, chlorine and bromine), an oxo group, analkanoyl group (preferably C_(1˜8)), an alkanoyloxy group (preferablyC_(1˜8)), an alkanoylamino group (preferably C_(1˜8)), carboxyl group,an alkoxycarbonyl group (preferably C_(2˜8)), a haloalkylcarbonyl group(preferably C_(2˜8)), an alkoxy group (preferably C_(1˜8)), a haloalkoxygroup (preferably C_(1˜8)), an alkyl group (preferably C_(1˜20)), aminogroup, an alkylamino group (preferably C_(1˜8)), a dialkylamino group(preferably C_(2˜16)), a cyclic amino group, an alkylaminocarbonyl group(preferably C_(2˜8)), carbamoyl group, hydroxyl group, nitro group,cyano group, mercapto group, an alkylthio group (preferably C_(1˜8)), analkylsulfonyloxy group (preferably C_(1˜8)), an alkylsulfonylamino group(preferably C_(1˜8)) and phenyl group.

[0200] Further, the said aromatic ring may be substituted with theabove-mentioned optionally substituted open-chain or cyclic hydrocarbongroup. Preferably, carbon number(s) of the hydrocarbon group is/are 1 to8. The said open-chain hydrocarbon group may be interrupted by anintervening group such as —O—, —CO—, —COO—, —S—, —SO—, —SO₂—, —NH—,—NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—, —NR³—SO₂—, —Si— or phosphoryl group(where R³ has the same meaning as the above-mentioned definition).

[0201] Still further, the said aromatic ring may be substituted with asubstituent represented by the formula R¹⁰—Z—R¹¹— (in the formula, R¹⁰and R¹¹ may be the same or different and each is an optionallysubstituted open-chain or cyclic hydrocarbon group, an optionallysubstituted aryl group, an optionally substituted heterocyclic group oran optionally substituted condensed heterocycylic group; and Z is anintervening group).

[0202] The compounds represented by the formulae (5) to (7) have anasymmetric carbon atom and, therefore, two optical isomers can exist.Accordingly, the pharmaceutical according to the present invention maycontain one of the optical isomers only or may contain a racemate.

[0203] There is no particular limitation for the pharmacologicallyacceptable salts of the compounds represented by the formulae (5) to (7)and, to be more specific, there may be exemplified inorganic acid saltsuch as a salt with hydrochloric acid, phosphoric acid, hydrobromic acidand sulfuric acid; organic acid salt such as a salt with acetic acid,formic acid, propionic acid, fumaric acid, maleic acid, succinic acid,tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid,methanesulfonic acid and benzenesulfonic acid; inorganic base salt suchas sodium salt, potassium salt and ammonium salt; and organic base saltsuch as dimethylamine salt and cyclohexylamine salt.

[0204] With regard to the specific examples of the above-mentionedsubstituents in the present invention, the following substituents may beexemplified.

[0205] With regard to the above “alkanoyl group”, there may beexemplified formyl group, acetyl group, propionyl group, butyryl groupand pivaloyl group.

[0206] With regard to the above “alkanoyloxy group”, there may beexemplified formyloxy group, acetoxy group, propionyloxy group,butyryloxy group and pivaloyloxy group.

[0207] With regard to the above “alkanoylamino group”, there may beexemplified acetylamino group, propionylamino group, butyrylamino groupand pivaloylamino group.

[0208] With regard to the above “alkoxycarbonyl group”, there may beexemplified methoxycarbonyl group, ethoxycarbonyl group, propoxycarbonylgroup, isopropoxycarbonyl group, butoxycarbonyl group, isobutoxycarbonylgroup, tert-butoxycarbonyl group and pentyloxycarbonyl group.

[0209] With regard to the above “haloalkylcarbonyl group”, there may beexemplified fluoroacetyl group, difluoroacetyl group, trifluoroacetylgroup, chloroacetyl group, dichloroacetyl group, trichloroacetyl group,bromoacetyl group, dibromoacetyl group, tribromoacetyl group,3-chloropropionyl group and 4-chlorobutyryl group.

[0210] The above “alkoxy group” means a straight or branched alkoxygroup and there may be exemplified methoxy group, ethoxy group, propoxygroup, isopropoxy group, butoxy group, tert-butoxy group, pentyloxygroup, tert-pentyloxy group and hexyloxy group.

[0211] The above “haloalkoxy group” means a group where halogen atom issubstituted in the above “alkoxy group” and there may be exemplifiedfluoromethoxy group, difluoromethoxy group, trifluoromethoxy group,chloromethoxy group, dichloromethoxy group, trichloromethoxy group,bromomethoxy group, dibromomethoxy group, tribromomethoxy group,iodomethoxy group, diiodomethoxy group, triiodomethoxy group,2-fluoroethoxy group, 2,2-difluoroethoxy group, 2,2,2-trifluoroethoxygroup, 2-chloroethoxy group, 2,2-dichloroethoxy group,2,2,2-trichloroethoxy group, 2-bromoethoxy group, 2,2-dibromoethoxygroup, 2,2,2-tribromoethoxy group, 3-chloropropoxy group and4-chlorobutoxy group.

[0212] The above “alkylamino group” means a group where an amino groupis substituted with an alkyl group and there may be exemplifiedmethylamino group, propylamino group, isopropylamino group, butylaminogroup, isobutylamino group, tert-butylamino group, pentylamino group,isopentylamino group, tert-pentylamino group and hexylamine group.

[0213] The above “dialkylamino group” means a group where an amino groupis disubstituted with an alkyl group in which the types of the alkylgroups may be the same or different and there may be exemplifieddimethylamino group, ethylmethylamino group, diethylamino group,methylpropylamino group, ethylpropylamino group, dipropylamino group,diisopropylamino group, dibutylamino group, diisobutylamino group,di-tert-butylamino group, dipentylamino group, dilsopentylamino group,di-tert-pentylamino group and dihexylamino group.

[0214] The above “cyclic amino group” means a group where an amino groupis in a cyclic form where four- to eight-membered cyclic amino groupsare preferred and there may be exemplified azetidinyl group,pyrrolidinyl group and piperidino group as well as those having oxygenatom, sulfur atom or nitrogen atom as a hetero atom such as morpholinogroup, thiomorpholino group and piperazinyl group. The nitrogen atom of4-position of the piperazinyl group may bear a substituent such as alower alkyl group or an aryl group.

[0215] The above “alkylaminocarbonyl group” means a group where the“alkylamino” moiety is the already-mentioned “alkylamino group” andthere may be exemplified methylaminocarbonyl group, ethylaminocarbonylgroup, propylaminocarbonyl group, isopropylaminocarbonyl group,butylaminocarbonyl group, isobutylaminocarbonyl group,tert-butylaminocarbonyl group, pentylaminocarbonyl group,isopentylaminocarbonyl group, tert-pentylaminocarbonyl group andhexylaminocarbonyl group.

[0216] The above “alkylthio group” means a straight or branchedalkylthio group and there may be exemplified methylthio group, ethylthiogroup, propylthio group, isopropylthio group, butylthio group,tert-butylthio group, pentylthio group, tert-pentylthio group andhexylthio group.

[0217] The above “alkylsulfonyloxy group” means a straight or branchedalylsulfonyloxy group, and there may be exemplified methylsulfonyloxygroup, ethylsulfonyloxy group, propylsulfonyloxy group,isopropylsulfonyloxy group, butylsulfonyloxy group,tert-butylsulfonyloxy group, pentylsulfonyloxy group,tert-pentylsulfonyloxy group and hexylsulfonyloxy group.

[0218] The above “alkylsulfonylamino group” means a group where an aminogroup is substituted with a straight or branched alkylsulfonyl group andthere may be exemplified methylsulfonylamino group, ethylsulfonylaminogroup, propylsulfonylamino group, isopropylsulfonylamino group,butylsulfonylamino group, tert-butylsulfonylamino group,pentylsulfonylamino group, tert-pentylsulfonylamino group andhexylsulfonylamino group.

[0219] In a substituent represented by the formula R¹⁰—Z—R¹¹— (in theformula, R¹⁰ and R¹¹ may be the same or different and each is anoptionally substituted open-chain or cyclic hydrocarbon group, arylgroup, heterocyclic group or condensed heterocyclic group; and Z is anintervening group), examples of the intervening group are —O—, —CO—,—COO—, —S—, —SO—, —SO₂—, —NH—, —NR³—, —NH—CO—, —NR³—CO—, —NH—SO₂—,—NR³—SO₂—, —Si— are phosphoryl group where R³ has the same meaning asthe above-mentioned definition.

[0220] With regard to the above-mentioned substituent, specific examplesthereof are the following substituents.

[0221] (a) With regard to a substituent where the intervening group is—O—, there may be exemplified methoxymethyl group, ethoxymethyl group,ethoxyethyl group, propoxymethyl group, propoxyethyl group,isopropoxymethyl group, butoxymethyl group, butoxyethyl group,butoxypropyl group, tert-butoxymethyl group, tert-butoxyethyl group,pentyloxymethyl group, pentyloxyethyl group, pentyloxypropyl group,pentyloxybutyl group, tert-pentyloxymethyl group, tert-pentyloxyethylgroup, hexyloxymethyl group, hexyloxyethyl group, hexyloxypropyl group,hexyloxypropyl group, benzyloxymethyl group and phenoxymethyl group.

[0222] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0223] (b) With regard to a substituent where the intervening group is—CO—, there may be exemplified acetylmethyl group, acetylethyl group,acetylpropyl group, acetylbutyl group, acetylpentyl group, acetylhexylgroup, propionylmethyl group, butyrylmethyl group, isobutyrylmethylgroup, valerylmethyl group, isovalerylmethyl group, hexanoylmethyl groupand phenylacetylmethyl group.

[0224] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0225] (c) With regard to a substituent where the intervening group is—COO—, there may be exemplified acetoxymethyl group, acetoxyethyl group,acetoxypropyl group, acetoxybutyl group, acetoxypentyl group,acetoxyhexyl group, propionyloxymethyl group,tert-butyloxycarbonylmethyl group, 1-isobutyryloxyethyl group,1-cyclohexyloxycarbonylethyl group, benzyloxycarbonylmethyl group,phenoxycarbonylmethyl group and pivaloyloxymethyl group.

[0226] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0227] (d) With regard to a substituent where the intervening group is—S—, there may be exemplified methylthiomethyl group, methylthioethylgroup, methylthiopropyl group, methylthiobutyl group, methylthioheptylgroup, methylthiohexyl group, methylthioisobutyl group, ethylthiomethylgroup, propylthiomethyl group, butylthiomethyl group, heptylthiomethylgroup, hexylthiomethyl group, benzylthiomethyl group andphenylthiomethyl group.

[0228] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0229] (e) With regard to a substituent where the intervening group is—SO₂—, there may be exemplified methylsulfonylmethyl group,methylsulfonylethyl group, methylsulfonylpropyl group,methylsulfonylbutyl group, methylosulfonylheptyl group,methylsulfonylhexyl group, methylsulfonylisobutyl group,ethylsulfonylmethyl group, propylsulfonylmethyl group,butylsulfonylmethyl group, heptylsulfonylmethyl group,hexylsulfonylmethyl group, benzylsulfonylmethyl group andphenylsulfonylmethyl group.

[0230] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0231] (f) With regard to a substituent where the intervening group is—SO—, there may be exemplified methylsulfinylmethyl group,methylsulfinylethyl group, methylsulfinylpropyl group,methylsulfinylbutyl group, methylsulfinylheptyl group,methylsulfinylhexyl group, methylsulfinylisobutyl group,ethylsulfinylmethyl group, propylsulfinylmethyl group,butylsulfinylmethyl group, heptylsulfinylmethyl group,hexylsulfinylmerhyl group, benzylsulfinylmethyl group andphenylsulfinylmethyl group.

[0232] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0233] (g) The substituent where the intervening group is —NH— is acompound represented by the formula R¹⁰—NH—R¹¹— where R¹¹ is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group and an optionally substituted condensed heterocyclicgroup as mentioned already. Examples of R¹⁰—NH— are methylamino group,ethylamino group, propylamino group, isopropylamino group, butylaminogroup, isobutylamino group, tert-butylamino group, pentylamino group,isopentylamino group, tert-pentylamino group, hexylamino group, anilinogroup and benzylamino group.

[0234] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0235] (h) The substituent where the intervening group is —NR³— is acompound represented by the formula R¹⁰—NR³—R¹¹— where R¹¹ is anoptionally substituted open-chain or cyclic hydrocarbon group, anoptionally substituted aryl group, an optionally substitutedheterocyclic group and an optionally substituted condensed heterocyclicgroup as mentioned already.

[0236] Examples of R¹⁰—NR³— are dimethylamino group, ethylmethylaminogroup, diethylamino group, methylpropylamino group, ethylpropylaminogroup, dipropylamino group, diisopropylamino group, dibutylamino group,diisobutylamino group, di-tert-butylamino group, dipentylamino group,diisopentylamino group, di-tert-pentylamino group, dihexylamino group,dibenzylamino group and methylbenzylamino group.

[0237] The substituent R³ has the same meaning as the above-mentioneddefinition.

[0238] Preferred carbon number(s) of the said substituent is/are 1 to10.

[0239] (i) With regard to the substituent where the intervening group is—NH—CO—, —NR³—CO—, NH—SO₂— or —NR³—SO₂—, there may be exemplified acompound where an intervening group is changed to the above-mentionedone in the above-mentioned compound where the intervening group Z is—NH— or —NR³—.

[0240] (j) With regard to the substituent where the intervening group Zis —Si—, there may be exemplified methylsilylmethyl group,methylsilylethyl group, methylsilylpropyl group, methylsilylbutyl group,methylsilylheptyl group, methylsilylhexyl group, methylsilylisobutylgroup, ethylsilylmethyl group, propylsilylmethyl group, butylsilylmethylgroup, heptylsilylmethyl group, hexylsilylmethyl group,benzylsilylmethyl group and phenylsilylmethyl group.

[0241] (k) The substituent where the intervening group Z is a phosphorylgroup is represented by the formulae;

[0242] (in the formulae, the substituents R¹⁰, R¹¹ and R¹² may be thesame or different and each is an optionally substituted open-chain orcyclic hydrocarbon group, an optionally substituted aryl group, anoptionally substituted heterocyclic group or an optionally substitutedcondensed hetero cyclic group). There may be exemplifiedmethylphosphoryl group, dimethylphosphoryl group andmethylethylphosphoryl group. Carbon number(s) of such a substituentis/are preferably 1 to 20.

[0243] All of the above-mentioned compounds according to the presentinvention may be manufactured by known methods or methods similarthereto. Methods for the manufacture of the compounds of the presentinvention will be exemplified as follows.

[0244] In the case of the compound represented by the formula (5) whereR¹ is not hydrogen, it can be manufactured by cyclization of a compound,for example, represented by the formula (11)

[0245] (in the formula, R¹, R² and X² are the same as those definedalready; and W is a leaving group) by means of an intramolecular ringclosing reaction.

[0246] Here, preferred leaving groups are the leaving groups which areknown per se such as halogen, ester group, mercapto group, etc.

[0247] As to the condition for the intramolecular ring closure reaction,known conditions may be used. Thus, for example, heating is carried outin an organic solvent, preferably in toluene, at about 30 to 100° C. or,preferably, about 50 to 70° C. for about 5 to 20 hours or, preferably,about 8 to 15 hours.

[0248] After that, the solvent is usually removed. Removal of thesolvent may be carried out under reduced pressure or in vacuo dependingupon necessity.

[0249] If desired, purification may be carried out by known methods suchas chromatography or filtration to give the compound represented by theformula (5).

[0250] In the case of the compound represented by the formula (5) whereR¹ is hydrogen, a starting material where R¹ is a protective group suchas benzyl group is firstly prepared and then it is hydrolyzed by a knownmethod or by a method similar thereto.

[0251] Depending upon the type of the protective group represented byR¹, catalytic reduction may be carried out instead of hydrolysis.Condition for the catalytic reduction may follow the known reactioncondition and there is exemplified a method where the starting materialis brought into contact with hydrogen gas under ordinary or highpressure in the presence of a catalyst such as palladium-carbon,palladium hydroxide-carbon, platinum oxide or palladium black in anamount of about 3 to 20% by weight or, preferably, about 5 to 15% byweight.

[0252] If desired, purification may be carried out by known methods suchas chromatography or filtration to give the compound represented by theformula (5).

[0253] In the case of the compound (6) where R¹ is not hydrogen and thesubstituent X² is O, the aimed compound is manufactured by adding anucleophilic agent containing X¹ (definition of X¹ is the same asdefined above) such as (COCl)₂ when X¹ is Cl or by adding a cyanide whenX¹ is CN to a compound represented by the formula (12)

[0254] (in the formula, R¹ has the same meaning as defined already) inan organic solvent, preferably ether.

[0255] This reaction is believed to proceed in such a mechanism that anintramolecular ketalization firstly takes place and a nucleophilic agentattacks the resulting hydroxyl group.

[0256] After that, there is carried out an after-treatment such asremoval of the solvent. Removal of the solvent may be carried out eitherunder reduced pressure or in vacuo upon necessity.

[0257] If desired, purification may be carried out by known methods suchas chromatography or filtration to give the compound represented by theformula (6).

[0258] In the case of the compound (7) where R¹ is not hydrogen, thesubstituent X² is O and R⁵and R⁶ form a benzene ring together with thecarbon atoms bonded thereto, a compound where X¹ is OH is synthesizedby, for example, addition of 1,2-isochroman-1,3,4-trione to benzylalcohol and pyridine.

[0259] After that, a nucleophilic agent containing X¹ (definition of X¹is the same as defined above) to be manufactured such as (COCl)₂ isadded when X¹ is Cl or, when X¹ is CN, a cyanide is added to substitutethe OH group with the substituent X² to manufacture the aimed compound.

[0260] Such a reaction may be carried out according to conventionalmeans.

[0261] The compounds represented by the above-mentioned formulae (1) to(7) may be metabolized in vivo, for example, as follows. Suchmetabolites may be a pharmaceutical according to the present inventionas well.

[0262] (in the formulae, Bn is benzyl group and Et is ethyl group)

[0263] The following compound (7-2), (7-3), (7-5) or (7-6) may bemetabolized to a compound represented by the formula (7-1). In somecases, the following compound (7-2), (7-3) or (7-5) may also bemetabolized to a compound where an ester group is converted to acarboxyl group.

[0264] The pharmaceutical in accordance with the present invention maybe in a dosage form such as tablets which are, if desired, sugar-coatedor film-coated, capsules, elixirs or microcapsules and may beadministered orally. Further, the pharmaceutical according to thepresent invention may be a parenteral preparation represented byinjection such as an aseptic solution or suspension preparation withwater or other pharmaceutically acceptable liquid.

[0265] The above-mentioned preparations may be manufactured by a methodwhich is known per se.

[0266] The pharmaceutical according to the present invention may furthercontain other pharmacologically active component which is effective toprogressive lesion after organic damage.

[0267] The pharmaceutical according to the present invention may stillfurther contain additives which have been used in the related art suchas binders, disintegrating agents, fillers, antiseptic agents,stabilizers and flavors.

[0268] With regard to the additive which may be mixed with tablets orcapsules, there may be exemplified those which have been used in therelated art such as binders, disintegrating agents, fillers, antisepticagents, stabilizers and flavors. To be more specific, there may be usedbinders such as hydroxypropyl cellulose, hydroxypropyl methyl cellulose,Macrogol, gelatin, corn starch, tragacanth and gum arabic;disintegrating agents such as starch and carboxymethyl cellulosecalcium; fillers such as lactose, starch and crystalline cellulose;swelling agents such as corn starch, gelatin and alginic acid;lubricants such as magnesium stearate and talc; sweeteners such assucrose, lactose and saccharine; and flavors such as peppermint, oilderived from Gaultheria ovatifolia ssp. Adenothrix and cherry. When thedosage form is a capsule, it is also possible to add a liquid carriersuch as fat/oil in addition to the above additives.

[0269] With regard to an aqueous solution for injection, it is possibleto use a physiological saline and other isotonic solution containingglucose and other excipients such as D-sorbitol, D-mannitol and sodiumchloride. In that case, it is further possible to jointly use theappropriate solubilization aids such as alcohol (e.g., ethanol),polyalcohol (e.g., propylene glycol and polyethylene glycol) andnonionic surface-active agent (e.g., Polysorbate 80™ and HCO-50). In thecase of an oily liquid for injection, sesame oil, soybean oil, etc. maybe used. Solubilizing aids such as benzyl benzoate and benzyl alcoholmay be also used together therewith. It is also possible to compoundwith buffer such as phosphate buffer and sodium acetate buffer;anesthetizing agent such as benzalkonium chloride and procainehydrochloride; stabilizer (such as human serum albumin and polyethyleneglycol; preserving agent (antiseptic) such as chlorobutanol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, benzyl alcohol and phenol;antioxidant; etc. The pharmaceutical preparation such as injectionprepared as such is usually filled in appropriate ampoules.

[0270] Daily dose of the pharmaceutical of the present invention mayvary depending upon the type of the effective ingredient, diseases to betreated, route for administration, dosage form, etc. and is notdefinitely decided. Preferably however, it is about 0.1 to 100 mg/kg,more preferably about 1 to 50 mg/kg and it is alsopossible to be about0.5to 50 mg/kg. Those compounds are of low toxicity and are able to beadministered either orally or parenterally.

[0271] The compounds of the present invention represented by theabove-mentioned formulae (1) to (7) have immunosuppressive or fibrosisinhibiting action. To be more specific, the compound according to thepresent invention exhibits an effect of selectively suppressing theeffector macrophage expressed in the damaged tissue caused by organicdamager or immune disease whereby it is able to selectively exhibit animmunosuppressive action selectively to specific tissues. Further, it isable to effectively inhibit the progress or worsening of the disease bythe said effector macrophage whereby it specifically exhibits a fibrosisinhibiting action selectively to damaged tissues.

[0272] Accordingly, the pharmaceutical which contains the compound ofthe present invention represented by the above-mentioned formulae (1) to(7) is able to be used as an immunosuppressant or a fibrosis inhibitor.To be more specific, the pharmaceutical can be used for prevention ofonset or progress of rejection upon allogeneic or xenogeneic cell,tissue or organ transplantation, acute or chronic glomerular nephritis,interstitial nephritis or diabetes mellitus; therapy and/or preventionof complications such as diabetic nephropathy, diabetic retinopathy anddiabetic neuropathy; therapy and/or prevention of chronic pancreatitis,arteriosclerosis, arteriosclerotic restenosis, pulmonary fibrosis,dialytic amyloidosis, chronic hepatitis, cerebrospinal degenerativedisease, asthma, rheumatic arthritis, chronic pigmentary dermatitis,psoriasis, autoimmune chronic organic tissue damage, endotoxin shockreaction by bacterial toxin, systemic intravascular coagulation andcancer or metastasis thereof; and prevention and therapy of aids virusinfection. It may also be used as a substitute for steroidal therapeuticagents.

[0273] Daily dose of the above-mentioned pharmaceutical according to thepresent invention used as an immunosuppressants or a fibrosis inhibitormay vary depending upon the type of the effective ingredient, thedisease to be treated, route of administration, dosage form, etc. and isnot definitely decided. Preferably however, it is about 0.1 to 100mg/kg, more preferably about 1 to 50 mg/kg and it is also possible to beabout 0.5 to 50 mg/kg in terms of the compound represented by theformulae (1) to (7). Those compounds are of low toxicity and are able tobe administered either orally or parenterally.

[0274] As mentioned above, the pharmaceutical according to the presentinvention which is used as an immunosuppressant or a fibrosis inhibitormay contain other pharmacological components showing animmunosuppressive action or a fibrosis inhibiting action as mentionedabove. Further, it may be in various dosage forms as mentioned aboveand, still further, it may contain known additives depending upon theabove-mentioned dosage form.

[0275] In the following Examples, Bn means a benzyl group.

EXAMPLE 1 Manufacture of (RS)-(−)-α-methyl-2-naphthalene-methyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate

[0276]

[0277] To a solution prepared by dissolving 164 mg (0.6 mmol, 1equivalent) of 2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylicacid manufactured by a method mentioned in paragraph [0018], page 5 ofJapanese Patent Laid-Open No. 04/338,331 in 2.2 ml of ether at 0° C.were added 240 μl (2.7 mmol, 4 equivalents) of (COCl)₂ gradually andthen two drops of dimethylformamide (DMF) were added thereto. At thattime, discharge of gas was observed.

[0278] The reaction solution was allowed to stand for 1 hour withstirring at 0° C., ether was removed in vacuo and the product was driedin vacuo.

[0279] The product was dissolved in 2.5 ml of ether, then 130 mg (0.75mmol, 1.1 equivalents) of (S)-(−)-α-methyl-2-naphthalenemethanol and 8mg (0.07 mmol, 0.1 equivalent) of dimethylaminopyrrolidone (hereinafter,abbreviated as DMAP) were gradually added and, at the same time, 140 μl(1 mmol, 1.5 equivalents) of triethylamine were added thereto as well.

[0280] After the reaction, the product is dissolved in 20 ml of ether,the solution was washed with an aqueous solution of NaHCO₃ and theorganic phase was dried over MgSO₄.

[0281] The solvent was removed in vacuo and, as a result of the firstpurification by silica gel, 175 mg (yield: 74%) of the product wereobtained. By means of purification using silica gel for several timesthereafter, an optical isomer was separated from a mixture of twodiastereomers.

[0282] About the compound of the formula (3), i.e.(S)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate:

[0283] Thin-layer chromatography: R_(f)=0.56 (hexane/ether, 1:1 (v/v))T_(f)=97-98° C. [α]²³ _(D)=−113 (c=3 in CHCl₃) RMN ¹H (300 MHz; CDCl₃) dppm: 1.44 (3H, d, J=6.7 Hz); 2.47-2.8 (4H, m); 6.03 (1H, q, J=6.7 Hz);6.82-6.88 (2H, m); 7.03-7.09 (2H, m); 7.34-7.35 (1H, m); 7.49-7.51 (2H,m); 7.73 (1H, s); 7.8-7.9 (3H, m). RMN ¹³C (75 MHz; CDCl₃) dppm: 21.54;27.4; 33.1; 75.5; 105.3; 116 (d, ²J_(CF)=23 Hz); 120.5 (d , ³J_(CF)=7.2Hz); 123.8; 125.6; 126.6; 127.8; 128.2; 128.7; 133.1; 133.3; 137.2;150.4; 159.3 (d, ¹J_(CF)=244 Hz); 166.0; 174.0. IR (CsI) ν: 3423; 2981;1797; 1758; 1504; 1290; 1194; 1165; 1082; 1044; 914; 857; 822; 751 cm⁻¹.

[0284] About the compound of the formula (4), i.e.(R)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate:

[0285] Thin-layer chromatography: R_(f)=0.51 (hexane/ether, 1:1 (v/v))T_(f)=117-118° C. [α]²³ _(D)=−20 (c=3 in CHCl₃) RMN ¹H (300 MHz; CDCl₃)d ppm: 1.62 (3H, d, J=6.7 Hz); 2.49-2.87 (4H, m); 6.05 (1H, q, J=6.7Hz); 6.76-6.81 (2H, m); 6.82-7.1 (2H, m); 7.17 (1H, m); 7.5-7.6 (2H, m);7.65 (1H, s); 7.7-7.8 (3H, m). RMN ¹³C (75 MHz; CDCl₃) d ppm: 21.5;27.41; 33.2; 75.6; 116 (d, ²J_(CF)=23 Hz); 120.4 (d, ³J_(CF)=8.6 Hz);123.8; 125.8; 126.6; 127.7; 128.1; 128.6; 133.0; 136.9; 150.3; 159.5 (d,¹J_(CF)=255 Hz); 166.3; 174.0. IR (CsI) ν: 3423; 2981; 1797; 1758; 1504;1290; 1194; 1165; 1082; 1044; 914; 857; 822; 751 cm⁻¹.

EXAMPLE 2 Manufacture of(±)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylic acid

[0286] (a) Manufacture of(−)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylic acid

[0287] C₁₁H₉FO₅

[0288] Molecular weight=240.1

[0289] White solid

[0290] T_(f)=128° C.

[0291] To a solution of 98 mg (0.24 mmol, 1 equivalent) of(S)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate represented bythe formula (3-1) manufactured in Example 1 dissolved in 4 ml of ethylacetate and 8 drops of ethanol was added a Pd/C catalyst in an amount of10 parts by weight to 100 parts by weight of the above solution. Theresulting solution was washed with water several times, stirred at roomtemperature and subjected to a catalytic reduction using hydrogen for4hours. The product was dissolved in ethyl acetate, the catalyst wasremoved by filtering through Celite (manufactured by Johns ManvilleSales Co.) and the solvent was removed in vacuo. The product waspurified by means of a reverse phase silica gel chromatography (RP 18)(eluent: acetonitrile/water, 1:1 (v/v)). Acetonitrile was removed invacuo and an aqueous phase was freeze-dried. The present compound waswhite powder and 45 mg (yield: 78%) were prepared.

[0292] (b) (+)-2-(4-fluorophenoxy)-5-oxotetrahydrofuran-3-carboxylicacid

[0293] C₁₁H₉FO₅

[0294] Molecular weight=240.1

[0295] White solid

[0296] T_(f)=128° C.

[0297] (R)-(−)-α-methyl-2-naphthalenemethyl2-(4-fluoro-phenoxy)-5-oxotetrahydrofuran-2-carboxylate represented bythe formula (4-1) manufactured in Example 1 was subjected to a catalyticreduction with hydrogen in the same manner as above whereupon thepresent compound was selectively manufactured. The present compound waswhite powder and 16 mg (yield: 58%) were obtained.

[0298] Compound (1): [a]²³ _(D)=−101 (c=0.6 in MeOH) Compound (2): [a]²³_(D)=+116 (c=0.3 in MeOH) RMN ¹H (300 MHz; CDCl₃) d ppm: 2.63-2.84 (4H,m); 7.08-7.14 (4H, m). RMN¹³C (75 MHz; CDCl₃) d ppm: 26.7; 32.6; 105.7;115.4 (d, ²J_(CF)=23 Hz); 120.5 (d , ³J_(CF)=8.6 Hz); 150.9; 159.2 (d,¹J_(CF)=235 Hz); 168.4; 175.1 IR (CsI) ν: 3082; 1775; 1507; 1253; 1199;1041; 978; 830; 708 cm⁻¹

EXAMPLE 3 Manufacture of benzyl2-(4-fluorobenzyl)-5-oxotetrahydrofuran-2-carboxylate

[0299]

[0300] C₁₉H₁₇FO₄

[0301] Molecular weight=328.3

[0302] White crystals

[0303] T_(f)=70° C.

[0304] In the presence of Amberyst resin 15 (manufactured by Rohm & HaasCo.), a solution where 192 mg (0.4 mmol, 1 equivalent) of dibenzyl2-(4-fluorobenzyl)-2-hydroxypentane-1,5-dicarboxylate were dissolved in6 ml of anhydrous toluene was heated at 60° C. for 10 hours. This wasreturned to room temperature and filtered through cotton to remove theAmberyst resin 15. The solvent was removed in vacuo and the product wasseparated by chromatography (eluent: hexane/ethyl acetate, 8.5:2.5(v/v)). After purification by recrystallization from ether-hexane twice,the present compound was obtained as thin flaky crystals in an amount of99 mg (yield: 76%).

[0305] Thin-layer chromatography: R_(f)=0.24 (hexane/ethyl acetate, 7:3(v/v)) RMN ¹H (300 MHz; CDCl₃) d ppm: 2.15-2.52 (4H, m); 3.1 (1H, d,J=14.4 Hz); 3.3 (1H, d, J=14.4 Hz); 5.14 (1H, d, J=12 Hz); 5.18 (1H, d,J=12 Hz); 6.92 (2H, t, J=8.6 Hz); 7.1 (2H, t, J=8.6 Hz); 7.25-7.36 (5H,m). RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.8; 30.4; 41.6; 67.6; 86.1; 115.2(d, ²J_(CF)=22 Hz); 128.3; 128.6; 129.5; 131.9 (d, ³J_(CF)=6.8 Hz);134.7; 162.3 (d, ¹J_(CF)=244 Hz); 170.8; 175.3. IR (CsI) ν: 1784; 1736;1508; 1223; 1189; 1097; 1056; 973; 910; 840; 700; 607 cm⁻¹. SM (IC/NH₃)m/z (intensite relative): 346=100% (MNH₄ ⁺); 347=23%; 674=19% (2×M+NH ₄⁺).

EXAMPLE 4 Manufacture of2-(4-fluorobenzyl)-5-oxo-tetrahydrofuran-2-carboxylic acid

[0306]

[0307] C₁₂H₁₁FO₄

[0308] Molecular weight=238.2

[0309] White crystals

[0310] T_(f)=93-94° C.

[0311] To a solution prepared by dissolving 71 mg (0.2 mmol, 1equivalent) of benzyl2-(4-fluorobenzyl)-5-oxo-tetrahydrofuran-2-carboxylate represented bythe formula (5-1), manufactured by the above Example 3, in a mixture of1.5 ml of ethyl acetate and 3 drops of ethanol was added a Pd/C catalystin an amount of 10 parts by weight to 100 parts by weight of the abovesolution. The reaction solution was washed with water several times andsubjected to a catalytic reduciton with hydrogen for 6 hours withstirring. Completion of the reaction was confirmed by means of athin-layer chromatography.

[0312] The product was filtered through Celite (manufactured by JohnsManville Sales Co.) and the solvent was evaporated in vacuo. As aresult, the present compound in white powder was obtained in an amountof 46 mg.

[0313] Thin-layer chromatography: R_(f)=0.23 (ethyl acetate/acetic acid,98:2 (v/v)) RMN ¹H (300 MHz; CDCl₃) d ppm: 2-2.55 (4H, m); 3.12 (H, d,J=14 Hz); 3.37 (1H, d, J=14 Hz); 7.01 (2H, t, J=8 Hz); 7.25 (2H, t, J=8Hz); 8.01 (1H, s). RMN ¹³C (75 MHz; CDCl₃) d ppm: 28.8; 31.9; 42.6;88.0; 115.7 (d, ²J_(CF)=21 Hz); 132.2; 133.3 (d, ³J_(CF)=7.2 Hz); 163.5(d, ¹J_(CF)=242 Hz); 174.2; 178.3. IR (CsI) ν: 3124; 1769; 1511; 1409;1270; 1244; 1223; 1178; 1037; 961; 933; 848; 773; 696; 658 cm⁻¹. SM(IC/NH₃) m/z (relative intensity): 256=100% (MNH₄ ⁺); 257=14%; 494=14%(2×M+NH₄ ⁺).

EXAMPLE 5 Manufacture of benzyl2-prop-2-ynyl-5-oxo-tetrahydrofuran-2-carboxylate

[0314]

[0315] C₁₅H₁₄O₄

[0316] Molecular weight=258.2

[0317] Colorless liquid

[0318] In the presence of Amberyst resin 15 (manufactured by Rohm & HaasCo.), a solution of 160 mg (0.43 mmol, 1 equivalent) of dibenzyl2-hydroxy-2-(2-prop-2-ynyl)pentane-1,5-dicarboxylate in 5 ml ofanhydrous toluene was stirred overnight at 60° C.

[0319] When the reaction finished, the reaction solution was returned toordinary temperature and spherical Amberyst resin was removed byfiltering through cotton. The solvent was removed in vacuo.

[0320] The product was purified by a silica type chromatography (eluent:hexane/ethyl acetate, 7:3 (v/v)). The present compound was colorlessliquid and was obtained in an amount of 85 mg (yield: 77%).

[0321] Thin-layer chromatography: R_(f)=0.23 (hexane/ethyl acetate, 7:3(v/v)) RMN ¹H (300 MHz; CDCl₃) d ppm: 2.08 (1H, t, J=2.6 Hz); 2.44-2.7(4H, m); 2.89 (1H, dd, J=17.3 Hz, J=2.6 Hz); 2.95 (1H, dd, J=17.3 Hz,J=2.6 Hz); 5.22 (1H, d, J=12 Hz); 5.28 (1H, d, J=12 Hz); 7.38 (5H, ml).RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.1; 28.1; 29.5; 67.8; 72.2; 77.0; 83.9;128.1; 128.6; 134.6; 169.8; 175.2. IR (CsI) ν: 3286; 1793; 1746; 1456;1419; 1339; 1261; 1170; 1068; 933; 753; 699 cm⁻¹. SM (IC/NH₃) m/z(intensite relative): 276=100% (MNH₄ ⁺); 277=19%; 534=16% (2×M+NH₄ ⁺).

EXAMPLE 6 Manufacture of benzyl2-benzyl-5-oxo-tetrahydrofuran-2-carboxylate

[0322]

[0323] C₁₉H₁₈O₄

[0324] Molecular weight=310.3

[0325] White crystals

[0326] T_(f)=93-94° C.

[0327] In the presence of Amberyst resin 15 (manufactured by Rohm & HaasCo.), a solution of 180 mg (0.43 mmol, 1 equivalent) of dibenzyl2-benzyl-2-hydroxypentane-1,5-dicarboxylate in 6 ml of anhydrous toluenewas stirred overnight at 60° C.

[0328] When the reaction finished, the reaction solution was returned toordinary temperature and spherical Amberyst resin was removed byfiltering through cotton. The solvent was removed in vacuo.

[0329] The product was purified by a silica type chromatography (eluent:hexane/ethyl acetate, 8:2 (v/v)). The present compound was a white solidand was obtained in an amount of 84 mg (yield: 63%).

[0330] Thin-layer chromatography: R_(f)=0.27 (hexane/ethyl acetate, 7:3(v/v)) RMN ¹H (300 MHz : CDCl₃) d ppm: 2.1-2.5 (4H, m); 3.15 (1H, d,J=14.3 Hz); 3.38 (1H, d, J=14.3 Hz); 5.2 (2H, s), 7.2-7.38 (10H, m). RMN¹³C (75 MHz; CDCl₃) d ppm: 27.9; 30.1; 42.3; 67.7; 86.2; 127.4; 128.3;128.5; 128.6; 128.7; 130.4; 171; 175.5. IR (CsI) ν: 1780; 1744; 1457;1432; 1269; 1174; 1082; 1040; 914 857; 758; 701; 604 cm⁻¹. SM (IC/NH₃)m/z (relative intensity): 328=100% (MNH₄ ⁺); 329=34.

EXAMPLE 7 Manufacture of 2-benzyl-5-oxotetrahydrofuran-2-carboxylic acid

[0331]

[0332] C12H12O4

[0333] Molecular weight=220.2

[0334] White crystals

[0335] T_(f)=110-111° C.

[0336] According to the manufacturing method for2-(3-fluorobenzyl)-5-oxotetrahydrofuran-2-carboxylic acid (Example 4),the present compound in white solid was obtained in an amount of 34 mg(yield: 90%) from benzyl 2-benzyl-5-oxotetrahydrofuran-2-carboylaterepresented by the formula (5-4) manufactured in the above Example 6.

[0337] Thin-layer chromatography: R_(f)=0.19 (ethylacetate/hexane/acetic acid, 6:4:0.2 (v/v/v)). RMN 1H (300 MHz; CDCl₃) dppm: 2.07-2.7 (6H, m); 3.16 (1H, d, J=14.3 Hz); 3.39 (1H, d, J=14.3 Hz);7.19-7.35 (5H, m); 7.88 (1H, sl). RMN ¹³C (75 MHz; CDCl₃) d ppm: 27.9;29.9; 42.2; 86.0; 127.5; 128.5; 130.5; 133.5; 175.5; 176.1. IR (CsI) ν:3032; 2938; 1782; 1712; 1185; 1082; 1044; 930; 746; 698 cm⁻¹. SM(IC/NH₃) m/z (relative intensity): 238=100% (MNH₄ ⁺); 239=15.

EXAMPLE 8 Manufacture of benzyl2-chloro-5-oxotetrahydrofuran-2-carboxylate

[0338]

[0339] C₁₂H₁₁ClO₄

[0340] Molecular weight=254.6

[0341] Yellow oil

[0342] To a solution of 7.5 g (31.7 mmol, 1 equivalent) of benzyl2-oxohemiglutarate dissolved in 105 ml of ether at 0° C. were added 8.3ml (98 mmol, 3 equivalents) of (COCl)₂ and then DMF (400 μl) was addedas well. At that time, discharge of gas was observed.

[0343] The mixture was stirred at 0° C. for 1 hour and then stirred atordinary temperature for 2 hours. The temperature was set at 0° C. andthe product was gradually neutralized with an aqueous solution of K₂CO₃.The product was extracted from ether (2×150 ml). The extracted organicphase was washed with water. After that, the reaction solution was driedover MgSO₄ and the solvent was removed in vacuo. The residue was addedto a column filled with silica gel and purified by passing an eluent ofhexane/ethyl (7:3 (v/v)) through the column to give 8 g of the presentinvention product as yellowish oil.

[0344] Thin-layer chromatography: R_(f)=0.4 (hexane/ethyl acetate, 7:3(v/v)) RMN ¹H (200 MHz; CDCl₃) d ppm: 2.48-2.89 (4H, m); 5.23 (2H, s);7.25-7.35 (5H, m). RMN¹³C (50 MHz; CDCl₃) d ppm: 26.6; 35.9; 68.6; 96.4;128.1; 128.6; 134.1; 164.6; 172.5. IR (CsI) ν: 1817; 1762; 1271; 1166;1088 cm⁻¹. SM (IC/NH₃) m/z (relative intensity): 255=13% (MH⁺); 272=100%(MNH₄ ⁺); 273=15%.

EXAMPLE 9 Manufacture of benzyl2-fluoro-5-oxotetrahydrofuran-2-carboxylate

[0345]

[0346] C₁₂H₁₁FO₄

[0347] Molecular weight=238.2

[0348] White crystals

[0349] T_(f)=57-58° C.

[0350] To a solution of 1 g (4.23 mmol, 1 equivalent) of benzyl2-oxohemiglutarate dissolved in 10 ml of methylene chloride was added asolution of 670 μl (5 mmol, 1.2 equivalents) ofdiethylaminotrifluorosulfonic acid (hereinafter abbrivated as DAST) in 4ml of methylene chloride at 0C. After mixing, the reaction solutionbecomes dark red and was placed in a darkroom of 4° C. for 48 hours. Theproduct was adsorbed on silica gel and separated by a silica typechromatography (eluent: hexane/ethyl acetate, 8.5:1.5 (v/v)). Thepresent compound was white crystalline and obtained in an amount of 725mg (yield: 72%).

[0351] Thin-layer chromatography: R_(f)=0.3 (hexane/ethyl acetate, 8:2(v/v)). RMN ¹H (300 MHz; CDCl₃) d ppm: 2.48-2.91 (4H, m); 5.3 (1H, d,J=12 Hz); 5.39 (1H, d, J=12 Hz); 7.44 (5H, sl). RMN ¹³C (75 MHz; CDCl₃)d ppm: 26.1; 30.6 (d, ²J_(CF)=26 Hz); 68.8; 11.2 (d, ¹J_(CF)=238 Hz);128.6; 128.9; 129.0; 134.2; 164.3 (d, ²J_(CF)=36 Hz); 173.0. IR (CsI) ν:1815; 1764; 1339; 1313; 1199; 1177; 1103; 1058; 973; 908; 776; 751; 699cm⁻¹. SM (IC/NH₃) m/z (relative intensity): 256=100% (MNH₄ ⁺); 257=12%.

[0352] Incidentally, the compound represented by the formula (6-5)

[0353] was able to be manufactured according to the method of Example 4from benzyl 2-fluoro-5-oxotetrahydrofuran-2-carboxylate manufactured inthis Example.

EXAMPLE 10 Manufacture of benzyl2-cyano-5-oxotetrahydrofuran-2-carboxylate

[0354]

[0355] C₁₂H₁₁FO₄

[0356] Molecular weight=238.2

[0357] White crystals

[0358] T_(f)=57-58° C.

[0359] To a solution where 200 mg (0.78 mmol, 1 equivalent) of benzyl2-chloro-5-oxotetrahydrofuran-2-carboxylate manufactured in Example 8was dissolved in 1.2 ml of tetrahydrofuran (THF) was added at −78° C. asolution of 209 mg (0.78 mmol, 1 equivalent) of cyanotetrabutylammoniumdissolved in 1.5 ml of THF. The reaction solution soon became red. Thiswas stirred at −78° C. for 15 minutes. The reaction solution wasreturned to ordinary temperature and gradually mixed with a mixture of 5ml of ether and 5 ml of water. After that, water was removed by ether(2×15 ml) and an organic phase was collected. The organic layer waswashed with water and dried over Na₂SO₄. After removal of the solvent,the residue was added to a column filled with silica gel and purified byadding an eluent hexane/ethyl acetate (7:3 (v/v)). The brown oilobtained at that time was further purified. The present compound was awhite solid and obtained in an amount of 177 mg (yield: 93%)

[0360] Thin-layer chromatography: R_(f)=0.3 (hexane/ethyl acetate, 7:3(v//v)) RMN ¹H (300 MHz; CDCl₃) dppm: 2.64-2.85 (4H, m); 5.35 (2H, s);7.4 (5H, ml). RMN ¹³C (75 MHz; CDCl₃) dppm: 25.9; 32; 69.6; 74.6; 114.6;128.3; 128.7; 129.0; 133.4; 163.8; 172.4. IR (CsI) ν: 3035; 1815; 1764;1498; 1456; 1417; 1379; 1271; 1158; 1072; 1048; 961; 895; 754; 698 cm⁻¹.SM (IC/NH₃) m/z (relative intensity): 263=100% (MNH₄ ⁺); 264=12%;280=24%.

EXAMPLE 12 Manufacture of benzyl1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate

[0361]

[0362] C₁₆H₁₂O₅

[0363] Molecular weight=284.2

[0364] White solid

[0365] T_(f)=77° C.

[0366] To a solution of 500 mg (2.84 mmol, 1 equivalent) of1,2-isochroman-1,3,4-trione dissolved in 6.5 ml of THF were added 292 μl(2.84 mmol, 1 equivalent) of benzyl alcohol and 230 μl (2.84 mmol, 1equivalent) of pyridine. The reaction solution which was yellow at firstbecame transparent after 15 minutes. The reaction solution was stirredat ordinary temperature for 2 hours.

[0367] After that, the solvent was removed in vacuo. The product wasdissolved again in a mixture of chloroform and water and, after that, anaqueous phase was extracted with chloroform (3×20 ml). The organic phasewas washed with 5% by volume of hydrochloric acid and with water. Theorganic phase was dried over Na₂SO₄ and the solvent was removed invacuo. The product was purified by chromatography using silica gel(eluent: hexane/ethyl acetate, 6:4 (v/v)). The present compound was awhite solid and obtained in an amount of 605 mg (yield: 75%).

[0368] Thin-layer chromatography: R_(f)=0.23 (hexane/ethyl acetate, 6:4(v/v) RMN ¹H (300 MHz; CDCl₃) d ppm: 5.24 (2H, s); 7.15-7.33 (1H, d,J=7.5 Hz); 7.64-7.76 (2H, m); 7.9 (1H, d, J=6.7 Hz). RMN ¹³C (75 MHz;CDCl₃) d ppm: 68.9; 122.8; 125.7; 126.8; 127.7; 128.5; 128.6; 131.3;133.9; 134.7; 144.6; 167.4; 167.8. IR (CsI) ν: 3384; 1746; 1467; 1277;1237; 1202; 1157; 1105; 1083; 907; 750; 694 cm⁻¹. SM (IC/NH₃) m/z(relative intensity): 302=100% (MNH₄ ⁺); 303=22%.

EXAMPLE 13 Manufacture of benzyl1-fluoro-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylate

[0369]

[0370] C₆H₁₁O₄

[0371] Molecular weight=302.7

[0372] White solid

[0373] T_(f)=78° C.

[0374] To a solution where 300 mg (1.05 mmol, 1 equivalent) of benzyl1-hydroxy-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate represented bythe formula (7-1) manufactured in the above Example 12 were dissolved in2 ml of methylene chloride was added at 0° C. a solution of 167 μl (1.22mmole, 1 equivalent) of DAST dissolved in 1.5 ml of methylene chloride.After addition, the reaction solution was stirred at 4° C. for 20 hours.The product was dissolved in methylene chloride, directly adsorbed onsilica gel and then purified by silica gel chromatography (eluent:hexane/ethyl acetate, 8:2 (v/v)). The present compound was colorless oiland obtained in an amount of 213 mg (yield: 71%).

[0375] Thin-layer chromatography: R_(f)=0.3 (hexane/ethyl acetate, 8:2(v/v)) RMN ¹H (300 MHz; CDCl₃) d ppm: 5.2 (1H, d, J=12 Hz); 5.3 (1H, d,J=12 Hz); 7.29-7.38 (5H, m); 7.68-7.81 (3H, m); 7.95-7.98 (1H, dd, J=6.7Hz, J=1.8 Hz). RMN ¹³C (75 MHz; CDCl₃) d ppm: 68.7; 107.2 (d,¹J_(CF)=238 Hz); 123.4; 126.1; 132.6; 135.4; 125.4; 128.2; 128.6; 128.8;142 (d, ²J_(CF)=20 Hz); 163.3 (d, ²J_(CF)=38.9 Hz); 165.7. IR (CsI) ν:3035; 1811; 1766; 1605; 1498; 1468; 1456; 1380; 1341; 1296; 1256; 1200;1163; 1129; 1107; 1083; 1046; 965; 905; 749; 688; 594 cm⁻¹. SM (IC/NH₃)m/z (relative intensity): 320=100% (MNH₄ ⁺); 305=25%.

EXAMPLE 14 Manufacture of benzyl1-chloro-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylate

[0376]

[0377] C₁₆H₁₁O₄Cl

[0378] Molecular weight=302.7

[0379] White solid

[0380] T_(f)=78° C.

[0381] Into 0.5 ml of ether were dissolved 50 mg (0.17 mmol, 1equivalent) of benzyl1-hydrodxy-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylate represented bythe formula (7-1) manufactured in the above Example 12. The solution wascooled at 0° C. and 80 μl (0.8 mmol, 5 equivalents) of (COCl)₂ and 3drops of DMF were added thereto. At that time, discharge of gas andprecipitate of white turbidity were observed. The reaction solution wasreturned to ordinary temperature and stirred for 3 hours. After thereaction, the product was dissolved in an aqueous solution of K₂CO₃, andthe aqueous phase was extracted with ether (3×10 ml). The organic phasewas washed with water and the solution was dried over Na₂SO₄. Thesolvent was removed in vacuo. There is no need to purify the presentcompound whereupon 51 mg of white solid were obtained.

[0382] Thin-layer chromatography: R_(f)=0.4 (hexane/ethyl acetate, 8:2(v/v)) RMN ¹H (300 MHz; CDCl₃) d ppm: 5.32 (2H, s); 7.35-7.38 (5H, m);7.66-7.95 (4 H, m). RMN ¹³C (75 MHz; CDCl₃) d ppm: 69.2; 91.9; 124;124.3; 125.8; 131.8; 135.5; 128.2; 128.7; 128.8; 134; 146.3; 163.8; 166.IR (CsI) ν: 3034; 1804; 1764; 1603; 1468; 1456; 1288; 1237; 1188; 1114;1042; 985; 960; 905; 746; 697 cm⁻¹. SM (IC/NH₃) m/z (relativeintensity): 286=100%; 320=95% (MNH₄ ⁺); 321=22%

EXAMPLE 15 Manufacture of benzyl3-oxo-1,3-dihydroisobenzofuran-1-carboxylate

[0383]

[0384] C₁₆H₁₂O₄

[0385] Molecular weight=268.2

[0386] White solid

[0387] T_(f)=101° C.

[0388] To a solution where 133 mg (0.43 mmol, 1 equivalent) of benzyl1-chloro-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate represented by theformula (7-3) manufactured in the above Example 14 were dissolved in 6ml of benzene were gradually added 122 μl (0.46 mmol, 1.05 equivalents)of Bu₃SnH and 4 mg (0.02 mmol, 0.05 equivalent) ofα,α′-azoisobutyronitrile (AIBN). The reaction solution was stirred at70° C. for 5 hours and then stirred overnight at ordinary temperature.After the reaction, the solvent was removed in vacuo. The product wasplaced in ether and washed with a 10% by volume of KF solution. Theorganic phase was dried over Na₂SO₄ and the solvent was removed invacuo. The residue was purified by a silica gel chromatography (eluent:hexane/ether, 7:3 (v/v)). The present compound was a white solid andobtained in an amount of 26 mg (yield: 23%).

[0389] Thin-layer chromatography: R_(f)=0.32 (hexane/ether, 6:4 (v/v))RMN ¹H (300 MHz; CDCl₃) d ppm: 5.17 (1H, d, J=12 Hz); 5.23 (1H, d, J=12Hz); 5.86 (1H, s); 7.3 (5H, m); 7.65-7.51 (3H, m); 7.83 (1H, d, J=7 Hz).RMN ¹³C (75 MHz; CDCl₃) d ppm: 68.0; 77.2; 122.7; 126.0; 130.2; 125.0;128.4; 128.7; 128.8; 134.5; 143.9; 166.5; 169.3. IR (CsI) ν: 3493; 3069;2966; 1780; 1758; 1601; 1468; 1455; 1378; 1320; 1281; 1256; 1215; 1197;1058; 1040; 949; 895; 755; 734; 697 cm⁻¹. SM (IC/NH₃) m/z (relativeintensity): 286=100% (MNH₄ ⁺); 287=227%, 554=17% (2×M+NH₄ ⁺).

EXAMPLE 16 Manufacture of benzyl1-(4-fluorophenoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carboxylate

[0390]

[0391] C₂₂H₁₅O₅

[0392] Molecular weight=378.34

[0393] White solid

[0394] T_(f)=75° C.

[0395] A solution where 450 mg (1.5 mmol, 1 equivalent) of benzyl1-hydroxy-3-oxo-1,3-dihydrobenzofuran-1-carboxylate represented by theformula (7-1) manufactured in the above Example 12 and 532 mg (4.75mmol, 3 equivalents) of 4-fluorophenol were dissolved in 2 ml ofmethylene chloride was heated at 65° C. for 5 minutes. After heating, itwas gradually added drop-by-drop to a solution where 355 mg (1.7 mmol, 1equivalent) of N,N-dicyclohexylcarbodiimide (DCC) were dissolved in 0.8ml of methylene chloride. After addition, the reaction solution washeated to reflux for 2.5 hours and the reaction container was returnedto ordinary temperature followed by diluting with 10 ml of methylenechloride. The precipitate was removed by filtration and the solvent wasremoved in vacuo. The product was purified by chromatography usingsilica gel (CH₂Cl₂/hexane, 6:4 (v/v)). The present compound was a whitesolid and obtained in an amount of 227 mg (yield; 40%).

[0396] Thin-layer chromatography: R_(f)=0.27 (CH₂Cl₂/hexane, 6:4 (v/v)).

[0397] RMN ¹H (300 MHz; CDCl₃) d ppm: 5.1 (1H, d, J=12 Hz); 5.2 (1H, d,J=12 Hz); 6.85-7.17 (4H, m); 7.28-7.34 (5H, m); 7.64-7.92 (4H, m). RMN¹³C (75 MHz; CDCl₃) d ppm: 68.5; 103.2; 115.9 (d, ²J_(CF)=23 Hz); 121.5(d, ³J_(CF)=8 Hz); 123.9; 125.8; 128.6; 131.9; 126.2; 128.2; 128.5; 134;134.9; 143.6; 149.4; 159.4 (d, ¹J_(CF)=242 Hz); 164.9; 166.9. IR (CsI)ν: 1790; 1747; 1603; 1506; 1465; 1272; 1258; 1205; 1097; 1040; 996; 959;830; 784; 741; 697 cm⁻¹. SM (IC/NH₃) m/z (relative intensity): 396=100%(MNH₄ ⁺); 397=33%.

EXAMPLE 17 Manufacture of1-(4-fluorophenoxy)-3-oxo-1,3-dihydro-isobenzofuran-1-carboxylic acid

[0398]

[0399] C₁₅H₉O₅

[0400] Molecular weight=288.2

[0401] White solid

[0402] T_(f)=143-147° C.

[0403] To a solution where 40 mg (0.1 mmol, 1 equivalent) of benzyl1-(4-fluorophenoxy)-3-oxo-1,3-dihydroisobenzofuran-1-carboxylaterepresented by the formula (7-5) manufactured in the above Example 16were dissolved in a mixture of 1.2 ml of ethyl acetate and 2 drops ofethanol was added a Pd/C catalyst in an amount of 10 parts by weight to100 parts by weight of the said solution. After the reaction solutionwas degassed several times, it was subjected to a catalytic reductionwith hydrogen with stirring at ordinary temperature for 1 hour. Thecatalyst was removed by filtering through Celite (manufactured by JohnsManville Sales Co.). The solvent was removed in vacuo. The compound wasa white solid and obtained in an amount of 28 mg.

[0404] Thin-layer chromatography: R_(f)=0.16 (ethylacetate/methanol/acetic acid, 95:0.5:0.1 (v/v/v)). RMN ¹H (300 MHz;CDCl₃) d ppm: 6.97-7.19 (4H, m); 7.70-7.88 (4H, m). RMN ¹³C (75 MHz;CDCl₃) d ppm: 105; 116.7(d, ²J_(CF)=23 Hz); 123.3 (d, ³J_(CF)=8.6 Hz);125.3; 126.4; 133.1; 136.3; 127.5; 145.5; 151.2; 160.9 (d, ¹J_(CF)=239Hz); 168.0; 168.2. IR (CsI) ν: 3448; 1797; 1734; 1501; 1466; 1257; 1188;1099; 1032; 960; 851; 783; 727 cm⁻¹. SM (IC/NH₃) m/z (relativeintensity): 306=100% (MNH₄ ⁺); 307=24% 594=23% (2×M+18).

EXAMPLE 18 Manufacture of the compound of the following formula

[0405]

[0406] C₉H₆O₅

[0407] Molecular weight=194.1

[0408] White solid

[0409] T_(f)=130-140° C.

[0410] Isochroman-1,3,4-trione (64mg, 0.36 mmol, 1 equivalent) wasdissolved in 0.5 ml of water followed by stirring overnight at ordinarytemperature. After that, the reaction solution was frozen and thenfreeze-dried. No purification was necessary and the present compound wasobtained white solid in an amount of 69 mg.

[0411] RMN ¹H (300 MHz; CDCl₃) d ppm: 7.61-7.91 (4H, m). RMN ¹³C (75MHz; CDCl₃) d ppm: 123.1 (sl); 125.1 (sl); 131.8; 134.6 (sl); 168.8(sl). IR (CsI) ν: 3494; 3067; 1777; 1745; 1467; 1386; 1285; 1232; 1197;1163; 1104; 1079; 1001; 930; 769; 706 cm⁻¹. n SM (IC/NH₃) m/z (relativeintensity): 212=100% (MNH₄ ⁺); 229=85%.

EXAMPLE 19 Manufacture of (RS)-(+)-α-methyl-2-naphthalene-methyl2-(4-fluorophenoxy)-5-oxotetrahydrofuran-2-carboxylate

[0412]

[0413] The above two compounds were manufactured by the same manner asin Example 1.

[0414] The above γ-lactone derivatives may be classified into thefollowing groups A-D when the conventional gamma-lactoneimmunosuppressive compound (a racemate of ethyl2-(4-fluorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate mentioned inJapanese Patent Laid-Open No. 04/338,331) is taken as a lead compound.

[0415] Group A: compounds where the conventional compound which isracemic is separated into single compounds which are optical isomers

[0416] Group B: compounds where oxocyclic oxygen of the lead compound issubstituted with carbon which are more stable to an enzymatic hydrolysisand show an activity similar to the lead compound.

[0417] Group C: compounds where 4-fluorophenoxy group of the leadcompound is substituted with an unstable substituent such as halogen,alkoxy group, benzoxy group, nitrile group, etc. Incidentally, suchcompounds may also be grasped as prodrugs of ketoglutaric acid havingactivity.

[0418] Group D: compounds where an aromatic ring is added to the leadcompound for stability of the compound and, in addition, 4-fluorophenoxygroup or halogen is introduced for affinity to lipid. Such compounds mayalso be grasped as a compound represented by the formula (7-1) havingactivity and prodrugs thereof.

[0419] (1) Evaluation of effector macrophage suppressive action showinga selective suppressive action to target cell damage in vitro

[0420] A suppressive effect of the test compound to effector macrophageinduction was investigated in vitro by an induction and productionsystem of spontaneous plaque-forming cell (SPEC) mentioned in M.Ishibashi, S. Jiang, Y. Kokado, S. Takahara, T. Sonoda:Immunopharmacologic effects of immunosuppressive agents explored by anew effector generation assay. Transplant Proc., 21:1854-1858, 1989.

TEST EXAMPLE 1

[0421] (Step 1: Separation of Human PBMC)

[0422] Heparin-added peripheral blood (40 ml) was collected from ahealthy person and the same amount of EDTA-added physiological salinewas added. PBMC were obtained by a specific gravity centrifugal methodusing Ficoll-Hypaque (manufactured by Pharmacia Fine Chemicals).Self-plasma was added thereto and allowed to stand at 37° C. for 10minutes and platelets were removed by a low-speed centrifugalseparation. RPMI 1640 liquid containing 15 μg/ml of gentamicin and 2 mML-glutamine was used for washing the PBMC. Concentration of the PBMC wasadjusted to 2×10⁶ cells/ml using the same liquid.

[0423] (Step 2: Addition of an Agonist and a Substance to be Tested)

[0424] Each 200 μl of a solution containing 2×10⁶ PBMC/ml prepared inthe step 1 were added to each well of a micro test plate,lipopolysaccharide (hereinafter, abbreviated as LPS) was diluted withthe RPMI 1640 liquid to make the final concentration 80 μg/ml and thesaid LPS solution was added to each well of the 200 μl-micro test plate.Further, each 50 μl of human serum of type AB were added so as to makethe concentration 10% by weight. Two such micro test plates wereprepared.

[0425] After that, to the above two micro test plates was added asolution where the test compound was diluted with 1% by weight of DMSOso as to make the concentration of the test compound 1 μM. Similarlywere prepared each two micro test plates where the final concentrationsof the test compound were 0.1 μM, 0.01 μM and 0.001 μM.

[0426] The culture liquid prepared as such was incubated for 6 days at37° C. in 5% CO₂.

[0427] (Step 3: Recovery of the Incubated PBMC)

[0428] The effector macrophage induced after the incubation was allrecovered using a rubber-policeman (spatula made of rubber). A Hankssolution containing 5 μg/ml of gentamicin was used for washing and,using the said solution, there was prepared a solution where theconcentration of the induced effector macrophage was 2×10⁶ per ml.

[0429] (Step 4: Preparation of Monolayered AutologousErythrocyte-Adhered Plate)

[0430] With regard to autologous erythrocytes, those which werepreserved at 4° C. in a phosphate physiological saline (hereinafter,abbreviated as PBS) to which 0.1% by weight of AB serum and autologouserythrocytes obtained from PBMC separation were used were used. Thepreserved autologous erythrocytes were washed for three times with aHanks solution to which no serum was added and made into a solution of4% by weight concentration using the said Hanks solution. Poly-L-lysinewas added to a Terasaki plate and washed with PBS every 20 minutes at37° C., the above-mentioned autologous erythrocyte solution was addedthereto immediately, the mixture was allowed to stand at 37° C. for 30minutes and the erythrocytes which were not adhered to the plate wereremoved to prepare a Terasaki plate to which self-erythrocytes in amonolayer were adhered.

[0431] A Hanks solution (4 μl) was added to the said Terasaki plate, 4μl of a solution containing the induced effect or macrophage obtained inthe step 3 were added thereto and the mixture was allowed to stand at37° C. for 2 hours. After completion of the reaction, it was fixed byformalin. Numbers of the produced SPFC which were adhered orphagocytized were measured.

[0432] (Step 5: Calculation of Total Amount of SPFC Produced)

[0433] A mean SPFC production number was calculated from the measuredvalues of SPFC production when the concentration of the test compoundwas the same. SPFC production number per 1×10⁶ of the induced effectormacrophage was calculated and, from the recovered induced effectormacrophage numbers, total SPFC production amount (S2) was calculated.

[0434] (Step 6: Judgment of Positive Compound Having the Activity)

[0435] As a control, no test compound was added and the same operationas above was carried out to calculate the total SPFC production amount(S1). The concentration of the test compound in which the total SPFCproduction amount when test compound was added (S2) was one half of thatwhen no test compound was added (S1) was defined as IC₅₀. The testcompound when its IC₅₀ was 1 μM or less was judged to be positive.

TEST EXAMPLE 2

[0436] The same operation as in Test Example 1 was carried out exceptthat, in the step 2, mitomycin-treated human PBMC (allogenic MLC;hereinafter, abbreviated as allo-MLC) was added to each well of themicro test plate so as to make its concentration 2×10⁶ per ml.

[0437] Here, the allo-MLC used was prepared in such a manner thatmitomycin was added to PBMC which was prepared by the same manner as inthe step 1 of Test Example 1 so as to make the concentration ofmitomycin 40 μg/ml followed by allowing to stand at 37° C. for 30minutes.

[0438] The result is shown in the following Table. The term “structurecharacteristic” in the Table means the above-mentioned groups A-D. Withregard to a functional classification of the compound, the compoundwhich selectively suppresses the SPFC production under the conditions ofallo-MLC stimulation or, in other words, the compound which selectivelysuppresses the induction of effector macrophage in the presence ofallo-MLC is classified as group I while the compound which selectivelysuppresses the SPFC production under the conditions of LPS stimulationor, in other words, the compound which selectively suppresses theinduction of effector macrophage in the presence of LPS is classified asgroup II. Further, the compound which selectively suppresses the SPFCproduction under the conditions of both allo-MLC stimulation and LPSstimulation or, in other words, the compound which non-selectivelysuppresses the induction of effector macrophage is classified as groupIII. Each of the “compound” in the Table is the compound bearing theabove-mentioned number. TABLE 1 SPFC Production SPFC ProductionInhibiting Inhibiting Concentration Concentration Func- Structure byall-MLC by LPS tional Charac- Stimulation Stimulation Classifi- teristicCompound (μM: IC₅₀) (μM: IC₅₀) cation A (1) 0.01 >1.0 Group I (2) >1.0 1Group II (3-2) >1.0 1 Group II (4-2) 0.01 >1.0 Group I B (5-2) 0.1 0.01Group II (5-4) 0.01 1 Group I C (6-1) >1.0 0.001 Group II (6-2) not done0.001 Group II (6-3) 0.1 0.001 Group II D (7-1) 0.1 1 Group I (7-2) >1.00.1 Group II (7-3) 0.01 >1.0 Group I (7-5) 0.01 >1.0 Group I (7-6) >1.00.01 Group II (7-7) >1.0 0.01 Group II Metabolite (9-1) 0.01 >1.0 GroupI Conventional Compound 0.1 0.1 Group III

[0439] In the Table, “Conventional Compound” is a racemic ethyl2-(4-fluorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate.

[0440] The optical isomer according to the present invention selectivelysuppressed the SPFC production by allo-MLC stimulation and that by LPSstimulation. The correlation in the biological activity depending uponthe structure as such was confirmed in the two types of optical isomersof “(1) and (2)” and “(3-2) and (4-2)”.

[0441] With regard to the compounds (group I) where SPFC production byallo-MLC stimulation is selectively suppressed, i.e., induction ofeffector macrophage in the presence of allo-MLC is selectivelysuppressed, the compounds represented by the formulae (1), (4-2), (5-4),(7-1), (7-3) and (7-5) correspondeded thereto. With regard to thecompounds (group II) where SPFC production by LPS stimulation isselectively suppressed, i.e., induction of effector macrophage in thepresence of LPS is selectively suppressed, the compounds represented bythe formulae (2), (3-2), (5-2), (6-1), (6-2), (6-3), (7-2), (7-6) and(7-7) corresponded thereto. There were some compounds where the SPFCproduction suppressive activity was from 10-fold to 100-fold as comparedwith the conventional compound.

[0442] (2) Evaluation of immunosuppressive action showing a selectivesuppressive effect on target cell damage in animal experiments

TEST EXAMPLE 3

[0443] Obstruction release model after complete obstruction ofunilateral ureter for 14 days

[0444] Experimental models were prepared by a method devised andestablished by Ishibashi (Michio Ishibashi, et al.: The Japanese Journalof Nephrology, 42:248, 2000) using male SD rats of 8-9 weeks age andabout 280 g. Thus, the rat was laparotomized under anesthetization withether and ureter was ligated with 7-0 Nylon at the height of the marginof lower pole of left kidney to close the abdomen. On the 14th day afterobstruction, the obstruction was released and urinary passage wasreconstructed using a cuff. Thus, after 14 days, the part of ligatedobstructed ureter was resected, a polyethylene tube of 25 gages(manufactured by Nippon Sherwood) was used as a cuff and inserted intoand retained at the lumen from the cut end of the lower normal ureter,then the cuff was also retained in the expanded upper ureter and each ofthem was ligated and fixed by 7-0 Nylon to reconstruct the urinarypassage. At the same time, the right kidney at the opposite side wasexcised. After the release of the obstruction, body weight was measured,blood was collected on the 2nd day, 5th day and 7th day from the releaseto measure serum creatinine, then the rat was sacrificed underanesthetization and the left obstruction-released kidney was excised.With regard to the excised kidney, there were carried out measurement ofweight of the kidney and pathological test for the kidney. When thecompound of the present invention was not administered to the model,destruction of renal structure was observed in pathological andmorphological investigations during the obstructed period and afterrelease of the obstruction with a lapse of time causing thickening ofglomerular Bowman's capsule wall, hyperplasia of mesangial cells,glomerular sclerosis, involution or dilation of urinary tubule, cellularinfiltration to interstitial tissues and fibrosis. Cellular infiltrationdid not increase the CD5-positive T cells and CD11b/CD18 (ED8)-positivemacrophage became dominant on the tenth day.

[0445] An in vivo biological test was carried out for the γ-lactonederivatives according to the present invention using the above models.With regard to the test compound, bulk powder of the test compound wasdissolved in an aseptic physiological saline together with gum arabicand the final concentration of gum arabic was 5% (vol/vol). Theconcentration of the test compound 30 mg/ml was suspended in 5% gumarabic-saline solution. The preparation was subcutaneously injected atthe dose of 30 mg/kg every day throughout the experiment. With regard tothe test compound, there were used (9-1), (4-2) and (7-3) as thecompounds of group I suppressing the SPFC production by allo-MLCstimulation, i.e., the compounds which selectively suppress theinduction of effector macrophage in the presence of allo-MLC while therewas used (3-2) as the compound of group II suppressing the SPFCproduction by LPS stimulation, i.e., the compounds which selectivelysuppress the induction of effector macrophage in the presence of LPS.Further, with regard to the compound of group III which suppresses theSPFC production by both allo-MLC stimulation and LPS stimulation, i.e.,the compound which non-selectively suppresses the induction of effectormacrophage, there was used a racemate of ethyl2-(4-flyuorophenoxy)-5-oxo-2-tetrahydrofurancarboxylate which is theknown compound.

[0446] The result is given in the following Table. In the Table,“functional classification” of the compound means the above group I,group II and group III. “Compound” means the compound bearing each ofthe above-mentioned compound numbers. The fact whether glomerular lesionwas suppressed was judged by the ameriolation of the lesions such asthickness of wall of Bowman's capsule, hyperplasia of mesangial cellsand glomerular sclerosis. The fact whether lesion of tubulointerstitialtissue was suppressed was judged by the ameriolation of the lesions suchas thickness of basement membrane of tubules, cellular infiltration andfibrosis of interstitial tissue. In the table, “control” is the resultwhen the same test as above was carried out using 5% gum arabic onlywhich is a solvent. TABLE 2 Serum Creatinine Rate of Rate of CasesWeight of Func- level (mg/dl) on Cases where where tubulo- kidney (g) ontional Case the 7th Day from Glomerular interstitial the 7th Day Classi-Com- Num- Release of Lesion was Lesion was from Release fication poundbers Obstruction Suppressed Suppressed of Obstruction Group (9-1) n = 32.0 ± 0.2 0% (0/4) 100% (4/4) 3.01 ± 0.56 I Control n = 3 2.0 ± 0.2 0%(0/3) 33% (1/3) 2.62 ± 0.70 (4-2) n = 4 2.7 ± 0.3 50% (2/4) 100% (4/4)3.94 ± 0.32 Control n = 4 2.7 ± 0.9 0% (0/4) 0% (0/4) 4.68 ± 0.66 (7-3)n = 4 2.0 ± 0.6 25% (1/4) 100% (4/4) 2.72 ± 0.47 Control n = 4 2.7 ± 0.925% (1/4) 25% (1/4) 2.69 ± 0.40 Group (3-2) n = 5 2.0 ± 0.4 100% (5/5)0% (5/5) 4.36 ± 0.95 II Control n = 3 2.6 ± 0.9 33% (1/3) 0% (0/3) 5.61± 1.08 Group Known n = 4 2.2 ± 0.2 75% (3/4) 75% (3/4) 2.73 ± 0.41 IIICompd Control n = 4 3.1 ± 0.8 0% (0/4) 0% (0/4) 2.21 ± 0.50

[0447] Both (9-1), (4-2) and (7-3) as the compounds of the group I whichsuppress the SPFC production by allo-MLC stimulation, i.e., thecompounds which selectively suppress the induction of effectormacrophage in the presence of allo-MLC and (3-2) as the compound of thegroup I which suppresses the SPFC production by LPS stimulation, i.e.,the compound which selectively suppresses the induction of effectormacrophage in the presence of SPFC showed a selective suppressiveeffect. Thus, the group I dominantly suppressed the lesion oftubulointerstitial tissue. The (3-2) of the group II did not suppressthe lesion of tubulointerstitial tissue but suppressed the glomerularlesion only. The conventional compound belongs to the group III and,although it showed suppression, no selective suppression was observed.

[0448] In order to further confirm whether the preventive andtherapeutic effect of the γ-lactone derivatives of the present inventionto the target cell damage is selective, (7-3) of the group I and (6-1)of the group II were used as test compounds and they were subcutaneouslyinjected at the dose of 30 mg/kg every day and compared with the controlgroup. With regard to the control group, only 5% gum arabic which is asolvent was used and the same test as above was carried out.

[0449] The result is shown in the following Table wherefrom it isapparent that, in all cases combined with group I and group IIcompounds, lesion of glomerulus and of tubulointerstitial tissue weresuppressed. TABLE 3 Rate of Weight Cases where of Kidney Creatinine Rateof Lesion of (g) on 7th level (mg/dl) Cases where Tubulo- Day fromCombination on 7th Day Glomerular interstitial Release of Case fromRelease Lesion was Tissue was of compounds Numbers of ObstructionSuppressed Suppressed Obstruction Combined n = 3 2.1 ± 0.3 100% (3/3)100% (3/3) 2.27 ± 0.16 use of (7-3) and (6-1) Control n = 5 2.5 ± 0.7 0%(0/5) 20% (1/5) 2.55 ± 0.45

TEST EXAMPLE 4 Evaluation Using Puromycin Chronic Nephrosis Model in Rat

[0450] Male SD rat of 8 weeks age of about 250 g was used and 50 μg/kgof puromycin were intravenously administered once according to a methodof Diamond, et al. (J. R. Diamond, I. Pesek, S. Ruggeri, M. J.Karnovsky: Essential fatty acid deficiency during acute puromycinnephrosis ameliorates late renal injury. Am. J. Physiol, 257:F798˜F807,1989). There was induced a chronic puromycin nephrosis rat wherealbuminuria increased from about tenth to twelfth week. After eighthweek from administration of puromycin, each of compound (9-1) andcompound (6-1) was subcutaneously injected at the dose of 30 mg/kg/dayeveryday until the 22nd week. Incidentally, as shown in the above Table1, (9-1) is a compound which suppresses the SPFC production by allo-MLCstimulation or, in other words, a compound which selectively suppressesthe induction of effector macrophage in the presence of allo-MLC (groupI) while (6-1) is a compound which suppresses the SPFC production by LPSstimulation or, in other words, a compound which selectively suppressesthe induction of effector macrophage in the presence of LPS (group II).

[0451] As a functional evaluation of kidney, albumin in urine wasmeasured every week using a kit for the measurement of albumin. On the22nd week, the rat was killed and anatomized to observe functional andmorphological changes of kidney.

[0452] The result is shown in the following Table. The compound (1-1)(group I) well suppressed the lesion of tubulointerstitial tissues whilethe compound (2-1) (group II) well suppressed the glomerular lesion.Functional average daily albuminuria was proportional to themorphological change.

[0453] Accordingly, it was found that the compounds of the group I whichsuppressed the induction of effector macrophage by allo-MLC stimulationselectively suppressed the lesion of tubulointerstitial tissues whilethe compounds of the group II which suppressed the induction of effectormacrophage by LPS stimulation selectively suppressed the glomerularlesion. TABLE 4 Rate of Rate of Cases where Func- Cases where Lesion oftioal Case Albumin in Glomerular Tubulointerstitial Classi- Com- Num-Urine Lesion was Tissue was fication pound bers (mg/day) SuppressedSuppressed Group I (9-1) n = 6 20, 7, 5, 5, 5, 4 50% (3/6) 83% (5/6)Group II (6-1) n = 6 56, 20, 11, 7, 4, 3 67% (4/6) 17% (1/6)

[0454] Correlation between the structural characteristic ofimmunosuppressive γ-lactone derivative and biological activities invitro and in vivo is given. Activity in vitro Activity in vitroInhibiting Concentration Inhibiting Concentration SuppressiveSuppressive Effect Structure- for SPEC Production by for SPEC Productionby Effect to to Lesion of Characteristic allo-MLC Stimulation LPSStimulation Glomerular Tubulointerstitial Experimental of compoundCompound (μM: IC₅₀) (μM: IC₅₀) Lesion Tissue Model of Rat A (3-2) >1.0 1∘ x UUO/Release (4-2) 0.01 >1.0 Δ ∘ UUO/Release C (6-1) >1.0 0.001 ∘ xCPN (6-2) not done 0.001 ∘ x UUO/Release D (7-3) 0.01 0.01 x ∘UUO/Release Metabolite (9) 0.01 >1.0 x ∘ UUO/Release ConventionalCompound 0.1 0.1 ∘ ∘ UUO/Release and CPN

[0455] As such, the γ-lactone derivatives in accordance with the presentinvention are able to selectively suppress the induction of effectormacrophage by allo-MLC stimulation and the induction of effectormacrophage by LPS stimulation. It has been also proved that, as a resultthereof, a suppressive effect depending upon the lesion is able to beachieved.

[0456] Accordingly, pharmaceutical preparations containing the γ-lactonederivatives according to the present invention can be applied dependingupon the lesions. When, for example, the progressive lesion after therenal damage comes to glomerulus in the case of renal diseases,administration of the pharmaceutical containing the compound of thegroup II is effective. On the other hand, when the progressive lesionafter the renal damage comes to tubulointerstitial tissue,administration of the pharmaceutical containing the compound of thegroup I is effective. When the progressive lesion after the renal damagecomes to both glomerulus and tubulointerstitial tissue, combination useof the pharmaceutical containing the compounds of the above-mentionedgroups I and II is effective. When the progressive lesion afterpancreatic damage comes to islets of Langerhans in the case of thepancreatic disease, administration of the pharmaceutical containing thecompound of the group II is effective while, when the progressive lesionafter pancreatic damage comes to exocrine, acinar and ductalinterstitial tissue of pancreas, administration of the pharmaceuticalcontaining the compound of the group I is effective. When theprogressive lesion after the renal damage comes to both islets ofLangerhans and exocrine, acinar and ductal interstitial tissue ofpancreas, combination use of the pharmaceutical containing the compoundsof the above-mentioned groups I and II is effective.

[0457] As mentioned above, the γ-lactone derivatives in accordance withthe present invention achieve a selective suppressive action unlike theconventional compounds. As a result of such an action, the γ-lactonederivatives in accordance with the present invention only suppress theactivation of induction of effector macrophage acting in a cytotoxicmanner to damaged organ tissue cells and do not suppress the activationof induction of macrophage participating in the regeneration of tissuesand, therefore, they are able to more effectively prevent or cure theprogressive lesion after the organic damage without lowering thedefensive ability of organism.

[0458] It has been also found that the compound (9-1), i.e. ethyl2-ketoglutarate, is one of the active metabolite derived from thecompounds of the group I which selectively suppress the SPFC productionby allo-MLC stimulation. Such an active metabolite shows an activity forsuppressing the lesion of in tubulointerstitial tissue in vivo.Similarly, the compound (9-2), i.e. benzyl 2-ketoglutarate, is one ofthe active metabolite of the group II which selectively suppresses theSPFC production by LPS stimulation and is able to suppress the lesion ofglomerulus in vivo.

TEST EXAMPLE 5 Acute and Chronic Toxicity Tests

[0459] Toxicity test of benzyl2-chloro-5-oxotetrahydrofuran-2-carboxylate represented by the formula(6-1) manufactured in Example 8 and ethyl 2-ketoglutarate represented bythe formula (9-1) was carried out. To be more specific, the former andthe latter were subcutaneously injected at the doses of 30 mg/kg/day and90 mg/kg/day, respectively for ten days and their toxicity wasinvestigated whereupon there was only noted an increase in weights ofthe liver and the spleen in a light degree.

[0460] When both were administered at the dose of 30 mg/kg/day forconsecutive 14 days, neither reduction of body weight nor death wasnoted and there was no abnormality at the subcutaneously injected areawhereupon they were found to be of low toxicity.

PREPARATION EXAMPLE 1

[0461] (Tablets) (1) Benzyl 2-chloro-5-oxotetrahydrofuran-2-  10 gcarboxylate (2) Lactose  90 g (3) Corn starch  29 g (4) Magnesiumstearate  1 g 130 g

[0462] The components (1) and (2) and 24 g of the component (3) weremixed with water to granulate, the resulting granules were mixed with 5g of the component (3) and the component (4), and the mixture wascompressed using a compressive tabletting machine to manufacture 1,000tablets of 7 mm diameter containing 10 mg of the component (1) pertablet.

PREPARATION EXAMPLE 2

[0463] (Capsules) (1) Benzyl 2-chloro-5-oxotetrahydrofuran-2- 50 mgcarboxylate (2) Lactose 14 mg (3) Corn starch 29 mg (4) Hydroxypropylcellulose  6 mg (5) Magnesium stearate  1 mg 100 mg per capsule

[0464] The above-mentioned components (1), (2), (3) and (4) were mixedand granulated according to a conventional method. The component (5) wasadded thereto and placed into a gelatin capsule by a conventional methodto give a capsule preparation.

Industrial Applicability

[0465] The present invention is able to provide a method for theinduction of effector macrophage which is induced and activatedcorresponding to the lesion inherent to the tissues after organic lesionand results in a progressive lesion after the organic damage.

[0466] By utilizing the said method, the present invention is able toprovide a method for screening compounds which are able to prevent,mitigate or cure the progressive lesion after organic damage such asglomerular lesion and tubulointerstitial lesion in the case of kidneyand, in the case of pancreas, exocrine interstitial lesion orpancreatitis and Langerhans islet lesion or diabetes. The presentinvention also provides a method for screening compounds which are ableto prevent, mitigate or cure diabetes and diabetic retinitis orpancreatitis and tubulointerstitial lesion complicated with pancreatitisat the same time.

[0467] It is also possible to provide a pharmaceutical for theprevention or the therapy of the above-mentioned progressive lesionafter the organic damage or a therapeutic method therefor by utilizingthe said screening method. The said pharmaceutical or the saidtherapeutic method has little side effect such as an unnecessarylowering of defense of organism or induction of new tissue damage and,in addition, it achieves repair and regeneration of the tissues withoutunnecessary lowering of the defense of organism whereby it is nowpossible to conduct the therapy for a long period on a continuous basisusing the said pharmaceutical or therapeutic method.

[0468] The novel γ-lactone derivatives according to the presentinvention are compounds which are able to selectively suppress theinduction of effector macrophage corresponding to the lesion inherent tothe tissues after the organic damage and are able to be used as theabove-mentioned pharmaceutical.

[0469] The novel γ-lactone derivatives according to the presentinvention have a selective induction-suppressive action for the aboveeffector macrophage and, as a result, they show a selectiveimmunosuppressive action or a fibrosis inhibiting action to specifictissues. Accordingly, the novel γ-lactone derivatives according to thepresent invention are able to be used not only as the above-mentionedpharmaceutical but also as an immunosuppressant or a fibrosis inhibitor.To be more specific, the pharmaceutical containing the novel γ-lactonederivative of the present invention is able to selectively suppress theprogress or worsening of the diseases to the target organ or the likeand, since its immunosuppressive action or fibrosis inhibiting action isstrong, it is effective for therapy and/or prevention of rejectionreaction upon of xenogenic or allogeneic cell, tissue or organtransplantation and endotoxin shock reaction by bacterial toxin,systemic intravascular coagulation, various inflammatory diseases,chronic inflammatory diseases and cancer. To be more specific, thepharmaceutical containing the novel γ-lactone derivative according tothe present invention is able to be used for prevention of onset orprogress of rejection reaction upon transplantation of allogeneic orxenogenic organs, cells or tissues, or acute or chronic glomerularnephritis and onset or progress of interstitial retinitis or diabetes;therapy and/or prevention of complications such as diabetic nephropathy,diabetic retinopathy and diabetic neuropathy; therapy and/or preventionof chronic pancreatitis, arteriosclerosis, arteriosclerotic restenosis,pulmonary fibrosis, dialytic amyloidosis, chronic hepatitis,cerebrospinal degeneration, asthma, rheumatic arthritis, chronicpigmentary skin diseases, psoriasis, autoimmune chronic organic tissuedamage, endotoxin shock reaction by cytotoxin, systemic intravascularcoagulation or cancer or its metastasis; and prevention and therapy ofinfection of AIDS virus. It is also able to be used as a substitute forsteroidal therapeutic agent.

1. A pharmaceutical composition, which comprises a compound suppressing the induction of effector macrophages.
 2. A preventive and therapeutic pharmaceutical selectively to progressive lesion of organic damages, which comprises a compound suppressing the induction of effector macrophages.
 3. A pharmaceutical for prevention and/or therapy of glomerular lesion of kidney, which comprises a compound suppressing the induction of effector macrophages caused by contact of human PBMC with lipopolysaccharide.
 4. A pharmaceutical for prevention and/or therapy of progressive tubulointerstitial lesion after renal damage, which comprises a compound suppressing the induction of effector macrophages caused by contact of human PBMC with mitomycin-treated human PBMC.
 5. A pharmaceutical for prevention and/or therapy of lesion of islet of Langerhans of pancreas, which comprises a compound suppressing the induction of effector macrophages caused by contact of human PBMC with lipopolysaccharide.
 6. A pharmaceutical for prevention and/or therapy of lesion of exocrine interstitial tissues of pancreas, which comprises a compound suppressing the induction of effector macrophages caused by contact of human PBMC with mitomycin-treated human PBMC.
 7. A pharmaceutical for prevention and/or therapy of diabetes mellitus and diabetic glomerular lesion, which comprises a compound suppressing the induction of effector macrophages caused by contact of human PBMC with lipopolysaccharide.
 8. A pharmaceutical for prevention and/or therapy of pancreatitis and lesion of interstitial tissues of urinary tubule which is a complication of pancreatitis, which comprises a compound suppressing the induction of effector macrophages caused by contact of human PBMC with mitomycin-treated human PBMC.
 9. A method for prevention and/or therapy of glomerular lesion of kidney, lesion of islets of Langerhans of pancreas or diabetes mellitus and diabetic glomerular lesion, which comprises using a pharmaceutical containing a compound suppressing the induction of effector macrophages caused by contact of human PBMC with lipopolysaccharide.
 10. A method for prevention and/or therapy of progressive tubulointerstitial lesion after renal damage, lesion of exocrine, acinar or ductal interstitial tissues of pancreas or pancreatitis, and lesion of interstitial tissues of urinary tubule, which comprises using a pharmaceutical containing a compound suppressing the induction of effector macrophages caused by contact of human PBMC with mitomycin-treated human PBMC.
 11. A method for screening a compound which is able to prevent, mitigate or treat glomerular lesion of kidney, lesion of islet of Langerhans of pancreas or diabetes mellitus and diabetic glomerular lesion, which comprises measuring a suppressive action of a compound to be tested against the induction of effector macrophage caused by contact of human PBMC with lipopolysaccharide.
 12. The method for screening according to claim 11, wherein effector macrophages are induced by incubation of human untreated PBMC in RPMI 1640 medium in the presence of a compound to be tested, lipopolysaccharide and human AB type serum, the said induced effector macrophages are brought into contact with monolayered autologous erythrocytes, and a compound showing less production of SPFC as compared with the absence of the said compound to be tested is screened.
 13. A method for screening a compound which is able to prevent, mitigate or treat progressive tubulointerstitial lesion after renal damage, lesion of exocrine interstitial tissues of pancreas or pancreatitis and lesion of interstitial tissues of urinary tubule, which comprises measuring a suppressive action of a compound to be tested against the induction of effector macrophages caused by contact of human PBMC with mitomycin-treated human PBMC.
 14. The method for screening according to claim 13, wherein effector macrophages are induced by a mixed incubation of mitomycin-treated human PBMC and human untreated PBMC in RPMI 1640 medium in the presence of a compound to be tested and human AB type serum, the said induced effector macrophages are brought into contact with monolayered autologous erythrocytes and a compound showing less production of SPFC as compared with the absence of the said compound to be tested is screened.
 15. A kit for screening a compound which is able to prevent, mitigate or treat glomerular lesion of kidney, lesion of islet of Langerhans of pancreas or diabetes mellitus and diabetic glomerular lesion, which comprises (a) human PBMC, (b) lipopolysaccharide, (c) human AB type serum, (d) RPMI 1640 medium and (e) a plate to which monolayered autologous erythrocytes are adhered.
 16. A kit for screening a compound which is able to prevent, mitigate or treat progressive tubulointerstitial lesion after renal damage, lesion of exocrine, acinar or ductal interstitial tissues of pancreas or pancreatitis and lesion of interstitial tissues of urinary tubule, which comprises (a) human PBMC, (b) mitomycin-treated human PBMC, (c) human AB type serum, (d) RPMI 1640 medium and (e) a plate to which monolayered autologous erythrocytes are is adhered.
 17. A method for the induction of effector macrophages which are a cause of glomerular lesion of kidney, lesion of islet of Langerhans of pancreas or diabetes mellitus and diabetic glomerular lesion, which comprises bringing lipopolysaccharide into contact with human PBMC.
 18. A method for the induction of effector macrophages which are a cause of progressive tubulointerstitial lesion after renal damage, lesion of exocrine interstitial tissues of pancreas or pancreatitis and lesion of interstitial tissues of urinary tubule, which comprises bringing human PBMC into contact with mitomycin-treated human PBMC.
 19. The pharmaceutical according to any of claims 1 to 8, which comprises a compound represented by the formula (1)

or a compound represented by the formula (2).


20. An optical isomer γ-lactone represented by the formula (3)

or by the formula (4)

(in the formula, R²¹ is an optionally substituted naphthyl group and R²² is an optionally substituted straight or branched hydrocarbon residue having 1 to 6 carbon atoms) or a mixture of the above optical isomers.
 21. The optical isomer γ-lactone or a mixture of the optical isomers according to claim 20, wherein R²¹ is naphthyl and R²² is methyl.
 22. A compound represented by the formula (5) or a pharmacologically acceptable salt thereof.

(in the formula, (a) R¹ and R² may be the same or different and each is hydrogen, an open-chain aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, an optionally substituted cyclic aliphatic hydrocarbon group, an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic group; X² is O, S or NR³ in which R³ is hydrogen, oxygen, an open-chain aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, an optionally substituted cyclic aliphatic hydrocarbon group, an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic group; and n is an integer from 1 to 5 or (b) X² is O, S or NR³; R¹, R² and R³ each is a substituent represented by the formula R¹⁰—Z—R¹¹— (in which R¹⁰ and R¹¹ may be the same or different and each is an optionally substituted open-chain or cyclic hydrocarbon group, an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic group; and Z is an intervening group); and n is an integer from 1 to 5).
 23. A compound represented by the formula (6) or a pharmacologically acceptable salt thereof.

(in the formula, R¹, X² and n have the same meaning as defined in claim 4; X¹ is halogen, cyano group, an optionally substituted mercapto group, an optionally substituted sulfo group, an optionally substituted sulfonyl group, an optionally substituted hydroxyl group, an optionally substituted amino group or an optionally substituted phosphoryl group).
 24. A compound represented by the formula (7) or a pharmacologically acceptable salt thereof.

(in the formula, R¹ and X² have the same meaning as defined in claim 4; X¹ has the same meaning as defined in claim 5; R⁵ and R⁶ may be the same or different and each is (a) hydrogen, a straight or branched aliphatic hydrocarbon group which may be substituted or interrupted by an intervening group, an optionally substituted cyclic aliphatic hydrocarbon group, an optionally substituted heterocyclic group, or an optionally substituted condensed heterocyclic group, (b) a substituent represented by the formula R¹⁰—Z—R¹¹— (in the formula, R¹⁰ and R¹¹ may be the same or different and each is an optionally substituted open-chain or cyclic hydrocarbon group, an optionally substituted aryl group, an optionally substituted heterocyclic group or an optionally substituted condensed heterocyclic group; and Z is an intervening group.), or (c) R⁵ and R⁶ together with the carbon atom to which they are attached form an optionally substituted aromatic ring).
 25. The pharmaceutical according to any of claims 1 to 8, which comprises a compound mentioned in any of claims 20 to
 24. 26. An immunosuppressant or a fibrosis inhibitor, which comprises a compound mentioned in any of claims 19 to
 24. 27. The pharmaceutical according to any of claims 1 to 8, which comprises ethyl 2-ketoglutarate or benzyl 2-ketoglutarate. 